BBA - Molecular Basis of Disease (v.1832, #12)
Editorial Board (i).
Amyloid beta-induced glycogen synthase kinase 3β phosphorylated VDAC1 in Alzheimer's disease: Implications for synaptic dysfunction and neuronal damage by P. Hemachandra Reddy (1913-1921).
Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase that is involved in the multiple signaling processes of a cell. Increasing evidence suggests that GSK3β plays a key role in multiple cellular processes in the progression of diabetes, obesity, Alzheimer's disease (AD), Parkinson's disease (PD), inflammatory diseases, schizophrenia, bipolar and several mood disorders, and mitochondrial diseases. Recent research has found that increased GSK3β activity is linked to the pathogenesis of AD through amyloid beta (Aβ), phosphorylated tau and mitochondrial dysfunction. Recent research has also revealed that GSK3β is elevated in AD-affected tissues and is critically involved in dissociating the voltage-dependent anion channel 1 (VDAC1) protein from hexokinases, and causing disrupted glucose metabolism, mitochondrial dysfunction and activating apoptotic cell death. The purpose of this article is to review recent research that is elucidating the role of GSK3β in AD pathogenesis. We discuss the involvement of GSK3β in the phosphorylation of VDAC1 and dissociation of VADC1 with hexokinases in AD neurons.
Keywords: Mitochondria; Amyloid beta; Amyloid beta precursor protein; Glycogen synthase kinase 3beta; Alzheimer's disease; Voltage-dependent anion channel 1;
Sphingosine kinase-1 inhibition protects primary rat hepatocytes against bile salt-induced apoptosis by Golnar Karimian; Manon Buist-Homan; Martina Schmidt; Uwe J.F. Tietge; Jan Freark de Boer; Karin Klappe; Jan Willem Kok; Laurent Combettes; Thierry Tordjmann; Klaas Nico Faber; Han Moshage (1922-1929).
Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (− 70%, P < 0.05) without inducing necrosis. GCDCA increased the S1P levels in hepatocytes (P < 0.05). GCDCA induced [Ca2 +] oscillations in hepatocytes and co-treatment with the [Ca2 +] chelator BAPTA repressed GCDCA-induced apoptosis. Ski-II inhibited the GCDCA-induced intracellular [Ca2 +] oscillations. Transcripts of all five S1P receptors were detected in hepatocytes, of which S1PR1 and S1PR2 appear most dominant. Inhibition of S1PR1, but not S1PR2, reduced GCDCA-induced apoptosis by 20%. Exogenous S1P also significantly reduced GCDCA-induced apoptosis (− 50%, P < 0.05), however, in contrast to the GCDCA-induced (intracellular) SphK1 pathway, this was dependent on S1PR2 and not S1PR1. Our results indicate that SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca2 +] signaling and activation of S1PR1.Schematic drawing summarizing the proposed mechanisms for glycochenodeoxycholic acid (GCDCA)-induced apoptosis in rat hepatocyte involving sphingosine kinase-1 (SphK1), sphingosine-1 phosphate (S1P) and sphingosine-1 phosphate receptors (S1PR1 and S1PR2). GCDCA induces the activation of SphK1 and increases the generation of endogenous S1P. Activation of SphK1 mediates GCDCA-induced [Ca2 +] oscillations in hepatocytes via generation of endogenous S1P and thereby induces apoptosis in hepatocytes. Exogenous S1P protects hepatocytes against GCDCA-induced apoptosis via S1PR2-dependent signaling. S1PR1-dependent signaling plays a minor role in GCDCA-induced apoptosis in rat hepatocytes. ASMase: acidic sphingomyelinase, Cdase: ceramidase, Ntcp: sodium-taurocholate cotransporting polypeptide, Ski II: SphK1 inhibitor, N: nucleus.Display Omitted
Keywords: Glycochenodeoxycholic acid; Sphingosine kinase; Calcium oscillation; Cholestatic liver disease; Sphingosine-1 phosphate; Sphingosine-1 phosphate receptor;
Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones by Jamal Elkharaz; Aslihan Ugun-Klusek; Dumitru Constantin-Teodosiu; Karen Lawler; R John Mayer; Ellen Billett; James Lowe; Lynn Bedford (1930-1938).
Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system, but the underlying mechanisms involved in neuroprotection and neurodegeneration remain unclear. Dysfunction of the ubiquitin proteasome system is one of the proposed hypotheses for the cause and progression of neuronal loss. We have performed quantitative two-dimensional fluorescence difference in-gel electrophoresis combined with peptide mass fingerprinting to reveal proteome changes associated with neurodegeneration following 26S proteasomal depletion in mouse forebrain neurones. Differentially expressed proteins were validated by Western blotting, biochemical assays and immunohistochemistry. Of significance was increased expression of the antioxidant enzyme peroxiredoxin 6 (PRDX6) in astrocytes, associated with oxidative stress. Interestingly, PRDX6 is a bifunctional enzyme with antioxidant peroxidase and phospholipase A2 (PLA2) activities. The PLA2 activity of PRDX6 was also increased following 26S proteasomal depletion and may be involved in neuroprotective or neurodegenerative mechanisms. This is the first in vivo report of oxidative stress caused directly by neuronal proteasome dysfunction in the mammalian brain. The results contribute to understanding neuronal–glial interactions in disease pathogenesis, provide an in vivo link between prominent disease hypotheses and importantly, are of relevance to a heterogeneous spectrum of neurodegenerative diseases.
Keywords: Neurodegeneration; Ubiquitin proteasome system; 26S proteasome; Oxidative stress; Peroxiredoxin 6; Astrocytes;
Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant human melanocortin-4 receptors by Xiu-Lei Mo; Ya-Xiong Tao (1939-1948).
The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor that plays an essential role in regulating energy homeostasis. Defects in MC4R are the most common monogenic form of obesity, with about 170 distinct mutations identified in human. In addition to the conventional Gs-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2). Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the Gs-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2-fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the Gs-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.
Keywords: Melanocortin-4 receptor; Inverse agonist; Biased signaling; Constitutive activity;
Acetylcholine leads to signal transducer and activator of transcription 1 (STAT-1) mediated oxidative/nitrosative stress in human bronchial epithelial cell line by Mirella Profita; Giusy Daniela Albano; Angela Marina Montalbano; Caterina Di Sano; Giulia Anzalone; Rosalia Gagliardo; Loredana Riccobono; Anna Bonanno; Liboria Siena; Michael Paul Pieper; Mark Gjomarkaj (1949-1958).
The induction of nitric oxide synthase (iNOS) expression via the signal transducer and activator of transcription 1 (STAT-1) is involved in the mechanism of oxidative/nitrosative stress. We investigated whether acetylcholine (ACh) generates oxidative/nitrosative stress in bronchial epithelial cells during airway inflammation of COPD and evaluated the effects of Tiotropium, a once-daily antimuscarinic drug, and Olodaterol, a long-acting β2-agonist on these mechanisms. Human bronchial epithelial cells (16-HBE) were stimulated (4 h, 37 °C) with induced sputum supernatants (ISSs) from healthy controls (HC) (n = 10), healthy smokers (HS) (n = 10) or COPD patients (n = 10), as well as with ACh (from 1 μM to 100 μM). The activation of STAT-1 pathway (STAT-1Ser727 and STAT-1Tyr701) and iNOS was evaluated in the cell lysates by Western blot analysis as well as nitrotyrosine levels by ELISA, while reactive oxygen species (ROS) were evaluated by flow cytometry. Finally, the effect of Tiotropium (Spiriva®) (100 nM), alone or in combination with Olodaterol (1 nM), was tested in this model. ISSs from COPD patients significantly increased the phosphorylation of STAT-1Ser727 and STAT-1Tyr701, iNOS and ROS/Nitrotyrosine when compared with ISSs from HC or HS subjects in 16-HBE cells. Furthermore, synthetic ACh increased all these parameters in stimulated 16HBE when compared with untreated cells. Tiotropium and Olodaterol reduced the oxidative/nitrosative stress generated by ACh and ISSs. We concluded that ACh mediated the oxidative/nitrosative stress involving the STAT-1 pathway activation in human bronchial epithelial cells during COPD. β2-Long acting and antimuscarinic drugs, normally used in the treatment of COPD as bronchodilator, might be able to control these cellular events.
Keywords: Epithelial cell; Oxidative/nitrosative stress; STAT-1; Acetylcholine;
Diabetes during pregnancy influences Hofbauer cells, a subtype of placental macrophages, to acquire a pro-inflammatory phenotype by Giorgia Sisino; Thomas Bouckenooghe; Sandra Aurientis; Pierre Fontaine; Laurent Storme; Anne Vambergue (1959-1968).
Growing evidence indicates that maternal pathophysiological conditions, such as diabetes, influence fetal growth and could program metabolic disease in adulthood. Placental cells, particularly Hofbauer cells (HBCs), which are placental macrophages characterized by an anti-inflammatory profile (M2), can sense the modified maternal environment. The goal of this study was to investigate the direct effect of hyperglycemia on HBCs. We studied, at mRNA and protein levels, some markers of M2 and M1 (pro-inflammatory) macrophages in placentae from control and diabetic patients to assess the balance between pro- and anti-inflammatory macrophages: an imbalance of M2 to M1 macrophages has been observed in humans. We used pregnant rats, receiving a single injection of streptozotocin (STZ), as a model of maternal diabetes. We noticed a M2-to-M1 macrophage unbalance as we observed in human. An in vitro model of isolated rat HBCs was used to identify the direct effects of high glucose. We found that high glucose stimulation activated genes belonging to TLR (Toll-Like Receptor)-dependent inflammatory pathways. Moreover, the HBCs stimulated by high glucose switched their M2 profile towards M1, with increased expression of pro-inflammatory cytokines and markers. We also noticed that the oxidative-stress pathway was activated in response to high glucose driven by Hif-1α. In this study, we demonstrated that diabetes/hyperglycemia affect the anti-inflammatory profile of HBCs, by stimulating these cells to acquire an inflammatory profile leading to adverse consequences for the fetal–placental–maternal axis.
Keywords: Maternal diabetes; Placenta; Hofbauer cells; High glucose; M1–M2 macrophages; Inflammation;
Egr1 regulates lithium-induced transcription of the Period 2 (PER2) gene by Se Hyun Kim; Hyun Sook Yu; Hong Guen Park; Yong Min Ahn; Yong Sik Kim; Young Han Lee; Kyooseob Ha; Soon Young Shin (1969-1979).
A growing body of evidence suggests that the circadian molecular system is involved in the pathogenic and therapeutic mechanisms underlying bipolar disorders. Lithium, a representative mood stabilizer, has been reported to induce the Period2 (PER2) gene; however, the underlying molecular mechanisms require further study. We found that lithium upregulated PER2 expression at the transcriptional level in neuronally differentiated SH-SY5Y human neuroblastoma cells. Promoter reporter analyses using serial deletions of the PER2 promoter revealed that two early growth response 1 (Egr1)-binding sites (EBS) between positions − 180 and − 100 are required for maximal activation of the PER2 promoter by lithium. Ectopic expression of Egr1 enhanced lithium-induced PER2 promoter activity, while a point mutation in EBS abolished it. Electrophoretic mobility shift assays and chromatin immunoprecipitation indicated that Egr1 bound directly to the PER2 promoter. Stimulation of the extracellular-signal regulated kinase (ERK)1/2/Elk1 pathway by lithium was functionally linked to PER2 expression through Egr1 induction, and lithium-induced PER2 expression was strongly attenuated by depletion of Egr1 by siRNA. Lithium also upregulated the expression of Per2 and Egr1 in mouse frontal cortex. Induction of Per2 by lithium was attenuated in Egr1 −/− mice. In conclusion, lithium stimulates PER2 transcription through the ERK/Elk1/Egr1 pathway in neuronal cells, indicating a connection between the ERK-Egr1 pathway and a circadian gene system in the mechanism of action of lithium.
Keywords: Bipolar disorder; Mood stabilizer; Lithium; Circadian gene; Per2; Egr1;
Phosphorylation and degradation of S6K1 (p70S6K1) in response to persistent JNK1 Activation by Jin Zhang; Zhanguo Gao; Jianping Ye (1980-1988).
S6K (ribosomal S6 kinase p70, p70S6K) activation requires phosphorylation at two stages. The first phosphorylation is independent of insulin stimulation and mediated by an unknown kinase. The second phosphorylation is mediated by mTOR in insulin dependent manner. In this study, we identified JNK1 (c-Jun N-terminal kinase 1) as a kinase in the first phosphorylation. S6K protein was phosphorylated by JNK1 at S411 and S424 in the carboxyl terminal autoinhibitory domain. The phosphorylation was observed in kinase assay with purified S6K as a substrate, and in cells after JNK1 activation by TNF-α or MEKK1 expression. The phosphorylation was detected in JNK2 null cells, but not in JNK1 null cells after TNF-α treatment. When JNK1 activation was inhibited by MKK7 knockdown, the phosphorylation was blocked in cells. The phosphorylation led to S6K protein degradation in NF-κB deficient cells. The degradation was blocked by inhibition of proteasome activity with MG132. In wide type cells, the phosphorylation did not promote S6K degradation when IKK2 (IKKβ, IκB kinase beta) was activated. Instead, the phosphorylation allowed S6K activation by mTOR, which stabilizes S6K protein. In IKK2 null cells or cells treated by IKK2 inhibitor, the phosphorylation led to S6K degradation. These data suggest that S6K is phosphorylated by JNK1 and the phosphorylation makes S6K protein unstable in the absence of IKK2 activation. This study provides a mechanism for regulation of S6K protein stability.
Keywords: Insulin resistance; Inflammation; Obesity; Liver; TNF-α;
Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction by Kyong-Jin Jung; Hee-Seong Jang; Jee In Kim; Sang Jun Han; Jeen-Woo Park; Kwon Moo Park (1989-1997).
Hydrogen sulfide (H2S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H2S in kidney fibrosis remains to be defined. Here, we investigated the role of H2S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H2S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H2S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H2S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H2S in UO-induced kidney fibrosis, suggesting that H2S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.
Keywords: Hydrogen sulfide; Fibrosis; Oxidative stress; NF-κB; TGF-β; Cystathionine β-synthase;
Activation of ERK accelerates repair of renal tubular epithelial cells, whereas it inhibits progression of fibrosis following ischemia/reperfusion injury by Hee-Seong Jang; Sang Jun Han; Jee In Kim; Sanggyu Lee; Joshua H. Lipschutz; Kwon Moo Park (1998-2008).
Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30 min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1 day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9 days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-β1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.
Keywords: Ischemia; Mitogen-activated protein kinase; Regeneration; Proliferation; Primary cilium; Exocyst;
Glutathione peroxidase contributes with heme oxygenase-1 to redox balance in mouse brain during the course of cerebral malaria by María Linares; Patricia Marín-García; Gabriela Martínez-Chacón; Susana Pérez-Benavente; Antonio Puyet; Amalia Diez; José M. Bautista (2009-2018).
Oxidative stress has been attributed both a key pathogenic and rescuing role in cerebral malaria (CM). In a Plasmodium berghei ANKA murine model of CM, host redox signaling and functioning were examined during the course of neurological damage. Host antioxidant defenses were early altered at the transcriptional level indicated by the gradually diminished expression of superoxide dismutase-1 (sod-1), sod-2, sod-3 and catalase genes. During severe disease, this led to the dysfunctional activity of superoxide dismutase and catalase enzymes in damaged brain regions. Vitagene associated markers (heat shock protein 70 and thioredoxin-1) also showed a decaying expression pattern that paralleled reduced expression of the transcription factors Parkinson disease 7, Forkhead box O 3 and X-box binding protein 1 with a role in preserving brain redox status. However, the oxidative stress markers reactive oxygen/nitrogen species were not accumulated in the brains of CM mice and redox proteomics and immunohistochemistry failed to detect quantitative or qualitative differences in protein carbonylation. Thus, the loss of antioxidant capacity was compensated for in all cerebral regions by progressive upregulation of heme oxygenase-1, and in specific regions by early glutathione peroxidase-1 induction. This study shows for the first time a scenario of cooperative glutathione peroxidase and heme oxygenase-1 upregulation to suppress superoxide dismutase, catalase, heat shock protein-70 and thioredoxin-1 downregulation effects in experimental CM, counteracting oxidative damage and maintaining redox equilibrium. Our findings reconcile the apparent inconsistency between the lack of oxidative metabolite build up and reported protective effect of antioxidant therapy against CM.
Keywords: Oxidative stress; Experimental cerebral malaria; Redox proteomics; Redox balance;
Down-regulation of LPCAT expression increases platelet-activating factor level in cirrhotic rat liver: Potential antiinflammatory effect of silybin by Eleonora Stanca; Gaetano Serviddio; Francesco Bellanti; Gianluigi Vendemiale; Luisa Siculella; Anna Maria Giudetti (2019-2026).
Cholestasis is one of the major causes of liver diseases. A chronic accumulation of toxic bile acids in the liver, which occurs in this condition, can induce fibrosis and cirrhosis. Inflammation is a fundamental component of acute and chronic cholestatic liver injury.Platelet-activating factor (PAF) is a proinflammatory lipid which may be generated by two independent pathways called the de novo and remodeling pathway being the last responsible for the synthesis of PAF during inflammation. In recent years a key role in PAF remodeling has been attributed to lysophosphatidylcholine acyltransferase (LPCAT) enzymes. Although the knowledge on their characteristic is growing, the exact mechanism of LPCAT in pathological conditions remains still unknown.Here, we reported that the level of lyso-PAF and PAF significantly increased in the liver of cirrhotic vs. control rats together with a significant decrease in both mRNA abundance and protein level of both LPCAT1 and LPCAT2. Acyltransferase activities of both LPCAT1 and LPCAT2 were parallel decreased in the liver of cirrhotic animals. Interestingly, treatment with silybin strongly decreased the level of both pro-inflammatory lipids and restored the activity and expression of both LPCAT1 and LPCAT2 of cirrhotic liver. Silybin effect was specific for LPCAT1 and LPCAT2 since it did not affect LPCAT3 mRNA abundance of cirrhotic liver.
Keywords: Biliary cirrhosis; Lysophosphatidylcholine acyltransferase; Platelet-activating factor; Silybin;
Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death by Sandra Dunning; Atta ur Rehman; Marjolein H. Tiebosch; Rebekka A. Hannivoort; Floris W. Haijer; Jannes Woudenberg; Fiona A.J. van den Heuvel; Manon Buist-Homan; Klaas Nico Faber; Han Moshage (2027-2034).
In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS.To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity.Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-peroxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE).Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1 mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE.Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death.
Keywords: Oxidative stress; Glutathione peroxidase; Catalase; Superoxide dismutase; Cell death;
Oral Porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3β signaling pathway by Makoto Ishikawa; Kaya Yoshida; Hirohiko Okamura; Kazuhiko Ochiai; Haruna Takamura; Natsumi Fujiwara; Kazumi Ozaki (2035-2043).
Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3β induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis.
Keywords: Periodontal disease; Diabetes mellitus; Porphyromonas gingivalis; Hepatic glycogenesis; SNAP26b;
A novel hypothesis for an alkaline phosphatase ‘rescue’ mechanism in the hepatic acute phase immune response by Adrianne F. Pike; Nynke I. Kramer; Bas J. Blaauboer; Willem Seinen; Ruud Brands (2044-2056).
The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.
Keywords: Alkaline phosphatase; Asialoglycoprotein receptor; Immunoglobulin G; Inflammation; Coagulation; Liver;
Differential expression and glycative damage affect specific mitochondrial proteins with aging in rat liver by Hilaire Bakala; Romain Ladouce; Martin A. Baraibar; Bertrand Friguet (2057-2067).
Aging is accompanied by the gradual deterioration of cell functions. Particularly, mitochondrial dysfunction, associated with an accumulation of damaged proteins, is of key importance due to the central role of these organelles in cellular metabolism. However, the detailed molecular mechanisms involved in such impairment have not been completely elucidated. In the present study, proteomic analyses looking at both changes at the expression level as well as to glycative modifications of the mitochondrial proteome were performed. Two-dimensional difference gel electrophoresis analysis revealed 16 differentially expressed proteins with aging. Thirteen exhibited a decreased expression and are crucial enzymes related to OXPHOS chain complex I/V components, TCA cycle or fatty acid β-oxidation reaction. On the other hand, 2 enzymes involved in fatty acid β-oxidation cycle were increased in aged mitochondria. Immunodetection and further identification of glycated proteins disclosed a set of advanced glycation endproduct-modified proteins, including 6 enzymes involved in the fatty acid β-oxidation process, and 2 enzymes of the TCA/urea cycles. A crucial antioxidant enzyme, catalase, was among the most strongly glycated proteins. In addition, several AGE-damaged enzymes (aldehyde dehydrogenase 2, medium chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA dehydrogenase) exhibited a decreased activity with age. Taken together, these data suggest that liver mitochondria in old rats suffer from a decline in their capacity for energy production, due to (i) decreased expression of OXPHOS complex I/V components and (ii) glycative damage to key fatty acid β-oxidation and TCA/urea cycle enzymes.
Keywords: Aging; Mitochondria; Proteomics; 2D-DIGE; Glycation; Protein expression;
Increased activation of the epidermal growth factor receptor in transgenic mice overexpressing epigen causes peripheral neuropathy by Maik Dahlhoff; Daniela Emrich; Eckhard Wolf; Marlon R. Schneider (2068-2076).
In the mammalian nervous system, axons are commonly surrounded by myelin, a lipid-rich sheath that is essential for precise and rapid conduction of nerve impulses. In the peripheral nervous system (PNS), myelin sheaths are formed by Schwann cells which wrap around individual axons. While the tyrosine kinase receptors ERBB2 and ERBB3 are established mediators of peripheral myelination, less is known about the functions of the related epidermal growth factor receptor (EGFR) in the regulation of PNS myelination. Here, we report a peripheral neurodegenerative disease caused by increased EGFR activation. Specifically, we characterize a symmetric and distally pronounced, late-onset muscular atrophy in transgenic mice overexpressing the EGFR ligand epigen. Histological examination revealed a demyelinating neuropathy and axon degeneration, and molecular analysis of signaling pathways showed reduced protein kinase B (PKB, AKT) activation in the nerves of Epigen-tg mice, indicating that the muscular phenotype is secondary to PNS demyelination and axon degeneration. Crossing of Epigen-tg mice into an EGFR-deficient background revealed the pathology to be completely EGFR-dependent. This mouse line provides a new model for studying molecular events associated with early stages of peripheral neuropathies, an essential prerequisite for the development of successful therapeutic interventions.
Keywords: Epigen; EGFR; Mouse model; Demyelination; Axon degeneration;
Ectopic calcification in β-thalassemia patients is associated with increased oxidative stress and lower MGP carboxylation by Federica Boraldi; Maria Garcia-Fernandez; Chiara Paolinelli-deVincenzi; Giulia Annovi; Leon Schurgers; Cees Vermeer; Paolo Cianciulli; Ivonne Ronchetti; Daniela Quaglino (2077-2084).
A number of beta-thalassemia (β-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from β-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of β-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from β-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from β-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.
Keywords: Elastin; Oxidative stress; Matrix Gla protein; Ectopic calcification; Beta-thalassemia;
Negative modulation of mitochondrial oxidative phosphorylation by epigallocatechin-3 gallate leads to growth arrest and apoptosis in human malignant pleural mesothelioma cells by Daniela Valenti; Lidia de Bari; Gabriella Arcangela Manente; Leonardo Rossi; Luciano Mutti; Laura Moro; Rosa Anna Vacca (2085-2096).
Increasing evidence reveals a large dependency of epithelial cancer cells on oxidative phosphorylation (OXPHOS) for energy production. In this study we tested the potential of epigallocatechin-3-gallate (EGCG), a natural polyphenol known to target mitochondria, in inducing OXPHOS impairment and cell energy deficit in human epitheliod (REN cells) and biphasic (MSTO-211H cells) malignant pleural mesothelioma (MMe), a rare but highly aggressive tumor with high unmet need for treatment. Due to EGCG instability that causes H2O2 formation in culture medium, the drug was added to MMe cells in the presence of exogenous superoxide dismutase and catalase, already proved to stabilize the EGCG molecule and prevent EGCG-dependent reactive oxygen species formation. We show that under these experimental conditions, EGCG causes the selective arrest of MMe cell growth with respect to normal mesothelial cells and the induction of mitochondria-mediated apoptosis, as revealed by early mitochondrial ultrastructure modification, swelling and cytochrome c release. We disclose a novel mechanism by which EGCG induces apoptosis through the impairment of mitochondrial respiratory chain complexes, particularly of complex I, II and ATP synthase. This induces a strong reduction in ATP production by OXPHOS, that is not adequately counterbalanced by glycolytic shift, resulting in cell energy deficit, cell cycle arrest and apoptosis. The EGCG-dependent negative modulation of mitochondrial energy metabolism, selective for cancer cells, gives an important input for the development of novel pharmacological strategies for MMe.
Keywords: EGCG; Malignant mesothelioma; Mitochondria; OXPHOS inhibition; STAT3;
Reduction of Nipbl impairs cohesin loading locally and affects transcription but not cohesion-dependent functions in a mouse model of Cornelia de Lange Syndrome by Silvia Remeseiro; Ana Cuadrado; Shimako Kawauchi; Anne L. Calof; Arthur D. Lander; Ana Losada (2097-2102).
Cornelia de Lange Syndrome (CdLS) is a genetic disorder linked to mutations in cohesin and its regulators. To date, it is unclear which function of cohesin is more relevant to the pathology of the syndrome. A mouse heterozygous for the gene encoding the cohesin loader Nipbl recapitulates many features of CdLS. We have carefully examined Nipbl deficient cells and here report that they have robust cohesion all along the chromosome. DNA replication, DNA repair and chromosome segregation are carried out efficiently in these cells. While bulk cohesin loading is unperturbed, binding to certain promoters such as the Protocadherin genes in brain is notably affected and alters gene expression. These results provide further support for the idea that developmental defects in CdLS are caused by deregulated transcription and not by malfunction of cohesion-related processes.
Keywords: Cornelia de Lange Syndrome; Nibpl; Cohesin; Transcription; Mouse model;
Combination of lipid metabolism alterations and their sensitivity to inflammatory cytokines in human lipin-1-deficient myoblasts by Caroline Michot; Asmaa Mamoune; Joseph Vamecq; Mai Thao Viou; Lu-Sheng Hsieh; Eric Testet; Jeanne Lainé; Laurence Hubert; Anne-Frédérique Dessein; Monique Fontaine; Chris Ottolenghi; Laetitia Fouillen; Karim Nadra; Etienne Blanc; Jean Bastin; Sophie Candon; Mario Pende; Arnold Munnich; Asma Smahi; Fatima Djouadi; George M. Carman; Norma Romero; Yves de Keyzer; Pascale de Lonlay (2103-2114).
Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha + Interleukin-1beta(TNF1α + IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.Figure: Working hypothesis for an experimental mechanistic link between lipin-1 deficiency and muscle lipid droplet formation along with an attempt for a clinical transposition in terms of predisposition and triggering of rhabdomyolysis episodes. Depicted events are either explicitly or implicitly supported by the present work. They are thought to confer muscle predisposition to lysis. Their experimental exacerbation by inflammation is put forward as a coherent trigger of muscle lysis.Display Omitted
Keywords: Rhabdomyolysis; Lipin-1; PAP1; ACACB; Lipid droplet; Inflammation;
Suppression of Aβ toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity by Antonina J. Kruppa; Stanislav Ott; Dhia S. Chandraratna; James A. Irving; Richard M. Page; Elena Speretta; Tiffany Seto; Luiz Miguel Camargo; Stefan J. Marciniak; David A. Lomas; Damian C. Crowther (2115-2126).
The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aβ toxicity.
Keywords: Puromycin-sensitive aminopeptidase; Amyloid; Alzheimer; Autophagy; Proteolysis;
Novel WWP2 ubiquitin ligase isoforms as potential prognostic markers and molecular targets in cancer by Surinder M. Soond; Paul G. Smith; Lloyd Wahl; Tracey E. Swingler; Ian M. Clark; Andrew M. Hemmings; Andrew Chantry (2127-2135).
The WWP2 E3 ubiquitin ligase has previously been shown to regulate TGFβ/Smad signalling activity linked to epithelial–mesenchymal transition (EMT). Whilst inhibitory I-Smad7 was found to be the preferred substrate for full-length WWP2-FL and a WWP2-C isoform, WWP2-FL also formed a stable complex with an N-terminal WWP2 isoform (WWP2-N) in the absence of TGFβ, and rapidly stimulated activating Smad2/3 turnover. Here, using stable knockdown experiments we show that specific depletion of individual WWP2 isoforms impacts differentially on Smad protein levels, and in WWP2-N knockdown cells we unexpectedly find spontaneous expression of the EMT marker vimentin. Re-introduction of WWP2-N into WWP2-N knockout cells also repressed TGFβ-induced vimentin expression. In support of the unique role for WWP2-N in regulating TGFβ/Smad functional activity, we then show that a novel V717M-WWP2 mutant in the MZ7-mel melanoma cell line forms a stable complex with the WWP2-N isoform and promotes EMT by stabilizing Smad3 protein levels. Finally, we report the first analysis of WWP2 expression in cancer cDNA panel arrays using WWP2 isoform-specific probes and identify unique patterns of WWP2 isoform abundance associated with early/advanced disease stages. WWP2-N is significantly downregulated in stage IIIC melanoma and up-regulated in stage II/III prostate cancer, and we also find isolated examples of WWP2-FL and WWP2-C overexpression in early-stage breast cancer. Together, these data suggest that individual WWP2 isoforms, and particularly WWP2-N, could play central roles in tumourigenesis linked to aberrant TGFβ-dependent signalling function, and also have potential as both prognostic markers and molecular therapeutic targets.
Keywords: TGFβ; Smads; Ubiquitin ligase; Transcription; Cancer; EMT;
Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis by Yang Woo Kwon; Soon Chul Heo; Geun Ok Jeong; Jung Won Yoon; Won Min Mo; Mi Jeong Lee; Il-Ho Jang; Sang Mo Kwon; Jung Sub Lee; Jae Ho Kim (2136-2144).
Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.
Keywords: Tumor necrosis factor-α; Mesenchymal stem cells; Endothelial progenitor cells; Angiogenesis; Ischemia;
Down-regulation of EBAF in the heart with ventricular septal defects and its regulation by histone acetyltransferase p300 and transcription factors smad2 and cited2 by Dongmei Su; Qian Li; Lina Guan; Xiaobo Gao; Huiling Zhang; Dandan E; Lei Zhang; Xu Ma (2145-2152).
As a NODAL pathway inhibitor, EBAF plays a critical role during mammalian cardiac development. As recent tests that have been conducted on gene-targeted mice indicate, its expression is frequently altered where cardiac defects are present. We aimed to explore the EBAF expression pattern and molecular mechanism of EBAF gene for VSD genesis. In this report, we show that the average expression of EBAF in the disease tissues of VSD patients was lower than the expression in normal fetuses without VSD. Further study showed that the expression pattern of EBAF was potentially involved in cardiomyocyte apoptosis by Annexin-V and RT-PCR assays. We also found that abnormal activation of NODAL-PITX2C pathway was associated with down-regulation of EBAF. By luciferase reporter assays, we find that EBAF expression is mediated by transcriptional factors smad2 and cited2. In addition, ChIP assays showed that histone acetyltransferase p300 is involved in the activation of EBAF through inducing hyperacetylation of histone H4 at the EBAF promoter. Co-immunoprecipitation also indicates that the expression of EBAF is regulated by a transcriptional complex including p300, smad2, and cited2. This study revealed a novel regulator mechanism of EBAF, which may be a potential molecular target for halting the onset of VSDs. They also indicate that smad2, cited2, and p300 may play important roles in modulating the confirmation of ventricular septal defects.
Keywords: EBAF; p300; smad2; cited2;
Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas by Rheal A. Towner; Nataliya Smith; Debra Saunders; Patricia Coutinho De Souza; Leah Henry; Florea Lupu; Robert Silasi-Mansat; Marilyn Ehrenshaft; Ronald P. Mason; Sandra E. Gomez-Mejiba; Dario C. Ramirez (2153-2161).
Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.Display Omitted
Keywords: Molecular magnetic resonance imaging; Glioma; Free radical; Immuno-spin-trapping; In vivo; Mouse;
Transcriptional modulation of pattern recognition receptors in acute colitis in mice by Bin Zheng; Mary E. Morgan; Hendrik J.G. van de Kant; Johan Garssen; Gert Folkerts; Aletta D. Kraneveld (2162-2172).
Pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), contribute to the development of intestinal inflammatory diseases, like inflammatory bowel disease (IBD). Supporting investigations of the underlying mechanisms of IBD, this study provides an extensive PRR expression survey together with T-cell associated factors along the murine colon during experimental colitis. 8–12 week-old C57BL/6 mice were treated with dextran sodium sulfate (DSS) to induce colitis. The mRNA expression levels of Tlr1–9, Nod1, Nod2, T cell subset-associated master transcription factors and cytokines were determined using qPCR. The expression of TLR2, 4, 5 and 6 was determined with immunohistochemistry. Th1 and Th17 associated responses were quantified in the mesenteric lymph nodes (mLNs) using flow cytometry. In DSS treated mice, the mRNA expression of the majority of PRRs was increased relative to healthy controls and correlated with the degree of inflammation. The exceptions were Tlr1 and Tlr5, which displayed unchanged and down-regulated transcription, respectively. Furthermore, in healthy animals, there was increased transcription of Tlr2, 3 and 5 near the caecum as opposed the region near the rectum. Within the inflamed regions, the mRNA expression of Th1-, Th17- and regulatory T-cell associated cytokines was enhanced, while there was no change for Th2-associated cytokines. In agreement with the mRNA expression, enhanced IFNγ and IL-17 producing cells were observed in stimulated mLNs. This study provides an extensive expression survey of PRRs along the colon during the acute colitis and shows that the induced inflammation is characterized by a Th1- and IL-17 mediated cytokine response.
Keywords: Inflammatory bowel disease; NOD; Th17 cells; Th1 cells; TLR;
Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions by Herman G.P. Swarts; Karl M. Weigand; Hanka Venselaar; Arn M. J.M. van den Maagdenberg; Frans G.M. Russel; Jan B. Koenderink (2173-2179).
Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the α2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase α2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the α and β subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na+ and increase the K+ affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K+ and Na+ affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).
Keywords: Na,K-ATPase; ATP1A2; Familial hemiplegic migraine; FHM2; α2 isoform; Domain interaction;
Spinal muscular atrophy: An update on therapeutic progress by Joonbae Seo; Matthew D. Howell; Natalia N. Singh; Ravindra N. Singh (2180-2190).
Humans have two nearly identical copies of survival motor neuron gene: SMN1 and SMN2. Deletion or mutation of SMN1 combined with the inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. SMA affects 1 in ~ 6000 live births, a frequency much higher than in several genetic diseases. The major known defect of SMN2 is the predominant exon 7 skipping that leads to production of a truncated protein (SMNΔ7), which is unstable. Therefore, SMA has emerged as a model genetic disorder in which almost the entire disease population could be linked to the aberrant splicing of a single exon (i.e. SMN2 exon 7). Diverse treatment strategies aimed at improving the function of SMN2 have been envisioned. These strategies include, but are not limited to, manipulation of transcription, correction of aberrant splicing and stabilization of mRNA, SMN and SMNΔ7. This review summarizes up to date progress and promise of various in vivo studies reported for the treatment of SMA.
Keywords: Spinal muscular atrophy; Survival motor neuron; Antisense oligonucleotide; Splicing; SMA therapeutics; Genetic disease therapy;
Activation of the endoplasmic reticulum stress response by the amyloid-beta 1–40 peptide in brain endothelial cells by Ana Catarina R.G. Fonseca; Elisabete Ferreiro; Catarina R. Oliveira; Sandra M. Cardoso; Cláudia F. Pereira (2191-2203).
Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1–40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca2 + homeostasis due to the release of Ca2 + from this intracellular store. Finally, Aβ1–40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1–40 concomitantly with caspase-12 activation. Furthermore, Aβ1–40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1–40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration.
Keywords: Alzheimer's disease; Amyloid-beta peptide; Endothelial cells; Endoplasmic reticulum stress; Calcium homeostasis; Apoptosis;
Met signaling in cardiomyocytes is required for normal cardiac function in adult mice by María Arechederra; Rita Carmona; María González-Nuñez; Álvaro Gutiérrez-Uzquiza; Paloma Bragado; Ignacio Cruz-González; Elena Cano; Carmen Guerrero; Aránzazu Sánchez; José Miguel López-Novoa; Michael D. Schneider; Flavio Maina; Ramón Muñoz-Chápuli; Almudena Porras (2204-2215).
Hepatocyte growth factor (HGF) and its receptor, Met, are key determinants of distinct developmental processes. Although HGF exerts cardio-protective effects in a number of cardiac pathologies, it remains unknown whether HGF/Met signaling is essential for myocardial development and/or physiological function in adulthood. We therefore investigated the requirement of HGF/Met signaling in cardiomyocyte for embryonic and postnatal heart development and function by conditional inactivation of the Met receptor in cardiomyocytes using the Cre-α-MHC mouse line (referred to as α-MHCMet-KO). Although α-MHCMet-KO mice showed normal heart development and were viable and fertile, by 6 months of age, males developed cardiomyocyte hypertrophy, associated with interstitial fibrosis. A significant upregulation in markers of myocardial damage, such as β-MHC and ANF, was also observed. By the age of 9 months, α-MHCMet-KO males displayed systolic cardiac dysfunction. Mechanistically, we provide evidence of a severe imbalance in the antioxidant defenses in α-MHCMet-KO hearts involving a reduced expression and activity of catalase and superoxide dismutase, with consequent reactive oxygen species accumulation. Similar anomalies were observed in females, although with a slower kinetics. We also found that Met signaling down-regulation leads to an increase in TGF-β production and a decrease in p38MAPK activation, which may contribute to phenotypic alterations displayed in α-MHCMet-KO mice. Consistently, we show that HGF acts through p38α to upregulate antioxidant enzymes in cardiomyocytes. Our results highlight that HGF/Met signaling in cardiomyocytes plays a physiological cardio-protective role in adult mice by acting as an endogenous regulator of heart function through oxidative stress control.
Keywords: Hepatocyte growth factor; Met; Cardiomyocytes; Oxidative stress; Heart; p38MAPK;
Endothelial dysfunction — A major mediator of diabetic vascular disease by Cristina M. Sena; Ana M. Pereira; Raquel Seiça (2216-2231).
The vascular endothelium is a multifunctional organ and is critically involved in modulating vascular tone and structure. Endothelial cells produce a wide range of factors that also regulate cellular adhesion, thromboresistance, smooth muscle cell proliferation, and vessel wall inflammation. Thus, endothelial function is important for the homeostasis of the body and its dysfunction is associated with several pathophysiological conditions, including atherosclerosis, hypertension and diabetes. Patients with diabetes invariably show an impairment of endothelium-dependent vasodilation. Therefore, understanding and treating endothelial dysfunction is a major focus in the prevention of vascular complications associated with all forms of diabetes mellitus. The mechanisms of endothelial dysfunction in diabetes may point to new management strategies for the prevention of cardiovascular disease in diabetes. This review will focus on the mechanisms and therapeutics that specifically target endothelial dysfunction in the context of a diabetic setting. Mechanisms including altered glucose metabolism, impaired insulin signaling, low-grade inflammatory state, and increased reactive oxygen species generation will be discussed. The importance of developing new pharmacological approaches that upregulate endothelium-derived nitric oxide synthesis and target key vascular ROS-producing enzymes will be highlighted and new strategies that might prove clinically relevant in preventing the development and/or retarding the progression of diabetes associated vascular complications.
Keywords: Endothelial dysfunction; Endothelium; Diabetes; Vascular function; Oxidative stress; Potential therapeutic target;
Fn14 in podocytes and proteinuric kidney disease by Maria Dolores Sanchez-Niño; Jonay Poveda; Ana Belen Sanz; Sergio Mezzano; Susana Carrasco; Beatriz Fernandez-Fernandez; Linda C. Burkly; Viji Nair; Matthias Kretzler; Jeffrey B. Hodgin; Marta Ruiz-Ortega; Rafael Selgas; Jesus Egido; Alberto Ortiz (2232-2243).
Non-proliferative proteinuric diseases are the most common primary glomerular disorders causing end-stage renal disease. These disorders may associate low level glomerular inflammation and podocyte expression of inflammatory mediators. However, the factors regulating podocyte expression of inflammatory mediators in vivo in non-immune disorders are poorly understood. We have now explored the regulation and role of TWEAK receptor Fn14 in mediating glomerular inflammation in cultured podocytes and in experimental and human non-immune proteinuria.Transcriptomics disclosed Fn14 and MCP-1 mRNA upregulation in glomeruli from patients with focal segmental glomerulosclerosis, as well as a correlation between the expression of both transcripts. Increased glomerular Fn14 and MCP-1 mRNA was confirmed in a second focal segmental glomerulosclerosis cohort and was also observed in membranous nephropathy. In human non-proliferative proteinuric kidney diseases podocytes displayed Fn14 and MCP-1 expression and NFκB activation. Podocyte Fn14 was increased in murine protein overload-induced proteinuria. In Fn14 knock-out mice with protein overload-induced proteinuria, glomerular and periglomerular macrophage infiltrates were reduced, as were MCP-1 mRNA and podocyte MCP-1 staining and podocyte numbers preserved as compared to wild-type counterparts. Adenovirus-mediated overexpression of TWEAK increased periglomerular macrophage infiltration in mice without prior kidney injury. In cultured podocytes inflammatory cytokines increased Fn14 mRNA and protein levels. TWEAK activated NFκB and increased MCP-1 mRNA and protein, an effect prevented by the NFκB inhibitor parthenolide. In conclusion, Fn14 activation results in NFκB-mediated pro-inflammatory effects on podocytes that may be relevant for the pathogenesis of non-proliferative proteinuric kidney disease of non-immune origin.Display Omitted
Keywords: Fn14; Focal segmental glomerulosclerosis; Kidney; Podocyte; Proteinuria; TWEAK;
Corrigendum to “Oct-1 recruitment to the nuclear envelope in adult-onset autosomal dominant Leukodystrophy” [Biochim. Biophys. Acta, Mol. Basis Dis. 1832 (3) (March 2013) 411–420] by Marta Columbaro; Elisabetta Mattioli; Nadir M. Maraldi; Michela Ortolani; Laura Gasparini; Maria Rosaria D'Apice; Diana Postorivo; Anna Maria Nardone; Sofia Avnet; Pietro Cortelli; Rocco Liguori; Giovanna Lattanzi (2244).
Effects of corticosterone and amyloid-beta on proteins essential for synaptic function: Implications for depression and Alzheimer's disease by Suthicha Wuwongse; Sally Shuk-Yee Cheng; Ginger Tsz-Hin Wong; Clara Hiu-Ling Hung; Natalie Qishan Zhang; Yuen-Shan Ho; Andrew Chi-Kin Law; Raymond Chuen-Chung Chang (2245-2256).
The relationship between Alzheimer's disease (AD) and depression has been well established in terms of epidemiological and clinical observations. Depression has been considered to be both a symptom and risk factor of AD. Several genetic and neurobiological mechanisms have been described to underlie these two disorders. Despite the accumulating knowledge on this topic, the precise neuropathological mechanisms remain to be elucidated. In this study, we propose that synaptic degeneration plays an important role in the disease progression of depression and AD. Using primary culture of hippocampal neurons treated with oligomeric Aβ and corticosterone as model agents for AD and depression, respectively, we found significant changes in the pre-synaptic vesicle proteins synaptophysin and synaptotagmin. We further investigated whether the observed protein changes affected synaptic functions. By using FM®4-64 fluorescent probe, we showed that synaptic functions were compromised in treated neurons. Our findings led us to investigate the involvement of protein degradation mechanisms in mediating the observed synaptic protein abnormalities, namely, the ubiquitin–proteasome system and autophagy. We found up-regulation of ubiquitin-mediated protein degradation, and the preferential signaling for the autophagic–lysosomal degradation pathway. Lastly, we investigated the neuroprotective role of different classes of antidepressants. Our findings demonstrated that the antidepressants Imipramine and Escitalopram were able to rescue the observed synaptic protein damage. In conclusion, our study shows that synaptic degeneration is an important common denominator underlying depression and AD, and alleviation of this pathology by antidepressants may be therapeutically beneficial.
Keywords: Alzheimer's disease; Depression; Synaptophysin; Synaptotagmin; Ubiquitin proteasome system; Antidepressant;
Structural plasticity of dendritic spines: The underlying mechanisms and its dysregulation in brain disorders by Kwok-On Lai; Nancy Y. Ip (2257-2263).
Dendritic spines are specialized structures on neuronal processes where the majority of excitatory synapses are localized. Spines are highly dynamic, and their stabilization and morphology are influenced by synaptic activity. This extrinsic regulation of spine morphogenesis underlies experience-dependent brain development and information storage within the brain circuitry. In this review, we summarize recent findings that demonstrate the phenomenon of activity-dependent structural plasticity and the molecular mechanisms by which synaptic activity sculpt neuronal connections. Impaired structural plasticity is associated with perturbed brain function in neurodevelopmental disorders such as autism. Information from the mechanistic studies therefore provides important insights into the design of therapeutic strategies for these brain disorders.
Keywords: Synaptic plasticity; NMDA receptor; Dendritic spine; BDNF; Rho GTPase; Local protein synthesis;
Green tea phenolics inhibit butyrate-induced differentiation of colon cancer cells by interacting with monocarboxylate transporter 1 by S. Sánchez-Tena; P. Vizán; P.K. Dudeja; J.J. Centelles; M. Cascante (2264-2270).
Diet has a significant impact on colorectal cancer and both dietary fiber and plant-derived compounds have been independently shown to be inversely related to colon cancer risk. Butyrate (NaB), one of the principal products of dietary fiber fermentation, induces differentiation of colon cancer cell lines by inhibiting histone deacetylases (HDACs). On the other hand, (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG), two abundant phenolic compounds of green tea, have been shown to exhibit antitumoral properties. In this study we used colon cancer cell lines to study the cellular and molecular events that take place during co-treatment with NaB, EC and EGCG. We found that (i) polyphenols EC and EGCG fail to induce differentiation of colon adenocarcinoma cell lines; (ii) polyphenols EC and EGCG reduce NaB-induced differentiation; (iii) the effect of the polyphenols is specific for NaB, since differentiation induced by other agents, such as trichostatin A (TSA), was unaltered upon EC and EGCG treatment, and (iv) is independent of the HDAC inhibitory activity of NaB. Also, (v) polyphenols partially reduce cellular NaB; and (vi) on a molecular level, reduction of cellular NaB uptake by polyphenols is achieved by impairing the capacity of NaB to relocalize its own transporter (monocarboxylate transporter 1, MCT1) in the plasma membrane. Our findings suggest that beneficial effects of NaB on colorectal cancer may be reduced by green tea phenolic supplementation. This valuable information should be of assistance in choosing a rational design for more effective diet-driven therapeutic interventions in the prevention or treatment of colorectal cancer.
Keywords: Epicatechin; Epigallocatechin gallate; Butyrate; Differentiation; Colon cancer;
Cardiac matrix: A clue for future therapy by Paras Kumar Mishra; Srikanth Givvimani; Vishalakshi Chavali; Suresh C. Tyagi (2271-2276).
Cardiac muscle is unique because it contracts ceaselessly throughout the life and is highly resistant to fatigue. The marvelous nature of the cardiac muscle is attributed to its matrix that maintains structural and functional integrity and provides ambient micro-environment required for mechanical, cellular and molecular activities in the heart. Cardiac matrix dictates the endothelium myocyte (EM) coupling and contractility of cardiomyocytes. The matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) regulate matrix degradation that determines cardiac fibrosis and myocardial performance. We have shown that MMP-9 regulates differential expression of micro RNAs (miRNAs), calcium cycling and contractility of cardiomyocytes. The differential expression of miRNAs is associated with angiogenesis, hypertrophy and fibrosis in the heart. MMP-9, which is involved in the degradation of cardiac matrix and induction of fibrosis, is also implicated in inhibition of survival and differentiation of cardiac stem cells (CSC). Cardiac matrix is distinct because it renders mechanical properties and provides a framework essential for differentiation of cardiac progenitor cells (CPC) into specific lineage. Cardiac matrix regulates myocyte contractility by EM coupling and calcium transients and also directs miRNAs required for precise regulation of continuous and synchronized beating of cardiomyocytes that is indispensible for survival. Alteration in the matrix homeostasis due to induction of MMPs, altered expression of specific miRNAs or impaired signaling for contractility of cardiomyocytes leads to catastrophic effects. This review describes the mechanisms by which cardiac matrix regulates myocardial performance and suggests future directions for the development of treatment strategies in cardiovascular diseases.
Keywords: Heart; MMP; TIMP; miRNA; Stem cell; Angiogenesis;
Gly161 mutations associated with Primary Hyperoxaluria Type I induce the cytosolic aggregation and the intracellular degradation of the apo-form of alanine:glyoxylate aminotransferase by Elisa Oppici; Alessandro Roncador; Riccardo Montioli; Silvia Bianconi; Barbara Cellini (2277-2288).
Primary Hyperoxaluria Type I (PH1) is a severe rare disorder of metabolism due to inherited mutations on liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme whose deficiency causes the deposition of calcium oxalate crystals in the kidneys and urinary tract. PH1 is an extremely heterogeneous disease and there are more than 150 disease-causing mutations currently known, most of which are missense mutations. Moreover, the molecular mechanisms by which missense mutations lead to AGT deficiency span from structural, functional to subcellular localization defects. Gly161 is a highly conserved residue whose mutation to Arg, Cys or Ser is associated with PH1. Here we investigated the molecular bases of the AGT deficit caused by Gly161 mutations with expression studies in a mammalian cellular system paired with biochemical analyses on the purified recombinant proteins. Our results show that the mutations of Gly161 (i) strongly reduce the expression levels and the intracellular half-life of AGT, and (ii) make the protein in the apo-form prone to an electrostatically-driven aggregation in the cell cytosol. The coenzyme PLP, by shifting the equilibrium from the apo- to the holo-form, is able to reduce the aggregation propensity of the variants, thus partly decreasing the effect of the mutations. Altogether, these results shed light on the mechanistic details underlying the pathogenicity of Gly161 variants, thus expanding our knowledge of the enzymatic phenotypes leading to AGT deficiency.Display Omitted
Keywords: Primary Hyperoxaluria; Alanine:glyoxylate aminotransferase; Pyridoxal phosphate; Hereditary metabolic disease; Protein aggregation; Protein misfolding;
Nuclear receptors expression chart in peripheral blood mononuclear cells identifies patients with Metabolic Syndrome by Simona D'Amore; Michele Vacca; Giusi Graziano; Andria D'Orazio; Marica Cariello; Nicola Martelli; Giuseppe Di Tullio; Roberto Salvia; Giuseppe Grandaliano; Anna Belfiore; Fabio Pellegrini; Giuseppe Palasciano; Antonio Moschetta (2289-2301).
Nuclear receptors are a class of 48 ligand-activated transcription factors identified as key players of metabolic and developmental processes. Most of these receptors are potential targets for pharmacological strategies in the Metabolic Syndrome. In the present study, we analyzed changes in the mRNA expression of nuclear receptors in the peripheral blood mononuclear cells of patients with Metabolic Syndrome, in order to identify novel biomarkers of disease and candidate targets for putative therapeutical approaches.We enrolled thirty healthy controls (14 M:16 F) and thirty naïve patients (16 M: 14 F; > 3 criteria for Metabolic Syndrome upon Adult Treatment Panel III) without organ damage. Using quantitative real-time PCR, we assessed the expression patterns of nuclear receptors in peripheral blood mononuclear cells. 33/48 nuclear receptors were expressed in peripheral blood mononuclear cells. In patients with Metabolic Syndrome, we found a significant down-regulation of the entire PPAR, NR4A and RAR families, together with a repression of RXRα, VDR, and Rev-Erbα. Furthermore, we performed a novel statistical analysis with classification trees, which allowed us to depict a predictive core of nuclear receptor expression patterns characterizing subjects with Metabolic Syndrome. Random Forest Analysis identified NOR1 and PPARδ, which were both reduced in peripheral blood mononuclear cells and specifically in CD14+ cells (mostly monocytes), as classifiers of Metabolic Syndrome, with high specificity and sensitivity.Our results point to the use of PPAR and NR4A mRNA levels in the overall peripheral blood mononuclear cells as biomarkers of Metabolic Syndrome and bona fide putative targets of pharmacological therapy.
Keywords: Nuclear receptors; Gene expression; Metabolic Syndrome; Inflammation; Epidemiology;
Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro by Roberta Cascella; Simona Conti; Benedetta Mannini; Xinyi Li; Joel N. Buxbaum; Bruno Tiribilli; Fabrizio Chiti; Cristina Cecchi (2302-2314).
Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aβ fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aβ42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aβ42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.
Keywords: Molecular chaperone; TTR protective effect; TTR-mediated oligomer clustering;
Tuning NF-κB activity: A touch of COMMD proteins by Paulina Bartuzi; Marten H. Hofker; Bart van de Sluis (2315-2321).
NF-κB is an important regulator of immunity and inflammation, and its activation pathway has been studied extensively. The mechanisms that downregulate the activity of NF-κB have also received a lot of attention, particularly since its activity needs to be terminated to prevent chronic inflammation and subsequent tissue damage. The COMMD family has been identified as a new group of proteins involved in NF-κB termination. All ten COMMD members share the structurally conserved carboxy-terminal motif, the COMM domain, and are ubiquitously expressed. They seem to play distinct and non-redundant roles in various physiological processes, including NF-κB signaling. In this review, we describe the mechanisms and proteins involved in the termination of canonical NF-κB signaling, with a specific focus on the role of the COMMD family in the down-modulation of NF-κB.
Keywords: Inflammation; NF-κB; COMMD family; Scaffold; Termination;
MitoQ, a mitochondria-targeted antioxidant, delays disease progression and alleviates pathogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis by Peizhong Mao; Maria Manczak; Ulziibat P. Shirendeb; P. Hemachandra Reddy (2322-2331).
Oxidative stress and mitochondrial dysfunction are involved in the progression and pathogenesis of multiple sclerosis (MS). MitoQ is a mitochondria-targeted antioxidant that has a neuroprotective role in several mitochondrial and neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Here we sought to determine the possible effects of a systematic administration of MitoQ as a therapy, using an experimental autoimmune encephalomyelitis (EAE) mouse model. We studied the beneficial effects of MitoQ in EAE mice that mimic MS like symptoms by treating EAE mice with MitoQ and pretreated C57BL6 mice with MitoQ plus EAE induction. We found that pretreatment and treatment of EAE mice with MitoQ reduced neurological disabilities associated with EAE. We also found that both pretreatment and treatment of the EAE mice with MitoQ significantly suppressed inflammatory markers of EAE, including the inhibition of inflammatory cytokines and chemokines. MitoQ treatments reduced neuronal cell loss in the spinal cord, a factor underlying motor disability in EAE mice. The neuroprotective role of MitoQ was confirmed by a neuron-glia co-culture system designed to mimic the mechanism of MS and EAE in vitro. We found that axonal inflammation and oxidative stress are associated with impaired behavioral functions in the EAE mouse model and that treatment with MitoQ can exert protective effects on neurons and reduce axonal inflammation and oxidative stress. These protective effects are likely via multiple mechanisms, including the attenuation of the robust immune response. These results suggest that MitoQ may be a new candidate for the treatment of MS.
Keywords: Mitochondrial dysfunction; Oxidative stress; Multiple sclerosis; Inflammation; Mitochondria-targeted antioxidant; Neuron;
Stress contributes to the development of central insulin resistance during aging: Implications for Alzheimer’s disease by Maite Solas; Bárbara Aisa; Rosa M. Tordera; María C. Mugueta; María J. Ramírez (2332-2339).
It is becoming evident that chronic exposure to stress not only might result in insulin resistance or cognitive deficits, but may also be considered a risk factor for pathologies such as depression or Alzheimer's disease (AD). There is great interest in determining the molecular mechanisms underlying interactions between stress, aging, memory and Alzheimer's disease (AD). We have used the chronic mild stress (CMS) model to study the effects of chronic stress on the aging process and the development of central insulin resistance and AD pathology. CMS aged mice showed cognitive impairments in the novel object recognition test. In addition, CMS aged mice displayed both peripheral insulin resistance, as shown by HOMA index, and decreased hippocampal levels of pIRS and downstream intracellular signaling (pAKT, pGSK and pERK1/2). Interestingly, there was a significant increase in both C99:C83 ratio and BACE1 levels in the hippocampus of CMS aged mice. Increased expression of the AD marker pTau was also found in stressed aged mice. Increased expression of the stress-activated protein kinase JNK was found in CMS aged mice, accompanied by significant decreases in glucocorticoid receptor (GR) expression and increases in mineralocorticoid receptor (MR) expression. It is suggested that the interaction of stress with aging should be considered when studying determinants of the onset and progression of AD.
Keywords: Cognitive deficit; Tau; Aβ; Glucocorticoid receptor; Mineralocorticoid receptor; JNK;
Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis by Manon Ruffin; Mélanie Voland; Solenne Marie; Monique Bonora; Elise Blanchard; Sabine Blouquit-Laye; Emmanuel Naline; Philippe Puyo; Philippe Le Rouzic; Loic Guillot; Harriet Corvol; Annick Clement; Olivier Tabary (2340-2351).
Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca2 +-activated Cl− channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl− channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl− channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.Display Omitted
Keywords: Anoctamin 1; TMEM16a; Cystic fibrosis; Airway; Cell proliferation; Bronchial epithelial repair;
LRRK2 guides the actin cytoskeleton at growth cones together with ARHGEF7 and Tropomyosin 4 by Karina Häbig; Sandra Gellhaar; Birgit Heim; Verena Djuric; Florian Giesert; Wolfgang Wurst; Carolin Walter; Thomas Hentrich; Olaf Riess; Michael Bonin (2352-2367).
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common genetic cause of Parkinson's disease (PD). However, LRRK2 function and molecular mechanisms causing the parkinsonian phenotype remain widely unknown. Most of LRRK2 knockdown and overexpression models strengthen the relevance of LRRK2 in regulating neurite outgrowth. We have recently identified ARHGEF7 as the first guanine nucleotide exchange factor (GEF) of LRRK2. This GEF is influencing neurite outgrowth through regulation of actin polymerization. Here, we examined the expression profile of neuroblastoma cells with reduced LRRK2 and ARHGEF7 levels to identify additional partners of LRRK2 in this process. Tropomyosins (TPMs), and in particular TPM4, were the most interesting candidates next to other actin cytoskeleton regulating transcripts in this dataset. Subsequently, enhanced neurite branching was shown using primary hippocampal neurons of LRRK2 knockdown animals. Furthermore, we observed an enhanced number of growth cones per neuron and a mislocalization and dysregulation of ARHGEF7 and TPM4 in these neuronal compartments. Our results reveal a fascinating connection between the neurite outgrowth phenotype of LRRK2 models and the regulation of actin polymerization directing further investigations of LRRK2-related pathogenesis.
Keywords: Actin cytoskeleton; Parkinson's disease; Growth cone; Neurite outgrowth;
RNA silencing of genes involved in Alzheimer's disease enhances mitochondrial function and synaptic activity by Maria Manczak; P. Hemachandra Reddy (2368-2378).
An age-dependent increase in mRNA levels of the amyloid precursor protein (APP), the microtubule-associated protein Tau, and voltage-dependent anion channel 1 (VDAC1) genes are reported to be toxic to neurons affected by Alzheimer's disease (AD). However, the underlying toxic nature of these genes is not completely understood. The purpose of our study was to determine the effects of RNA silencing of APP, Tau, and VDAC1 genes in AD pathogenesis. Using human neuroblastoma (SHSY5Y) cells, we first silenced RNA for APP, Tau, and VDAC1 genes, and then performed real-time RT-PCR analysis to measure mRNA levels of 34 genes that are involved in AD pathogenesis. Using biochemical assays, we also assessed mitochondrial function by measuring levels of H2O2 production, lipid peroxidation, cytochrome c oxidase activity, ATP production, and GTPase enzymatic activity. We found that increased mRNA expression of synaptic function and mitochondrial fission genes, and reduced levels of mitochondrial fusion genes in RNA silenced the SHSY5Y cells for APP, Tau and VDAC1 genes relative to the control SHSY5Y cells. In addition, RNA-silenced APP, Tau, and VDAC1 genes in SHSY5Y cells showed reduced levels of H2O2 production, lipid peroxidation, fission-linked GTPase activity, and increased cytochrome oxidase activity and ATP production. These findings suggest that a reduction of human APP, Tau, and VDAC1 may enhance synaptic activity, may improve mitochondrial maintenance and function, and may protect against toxicities of AD-related genes. Thus, these findings also suggest that the reduction of APP, Tau, and VDAC1 mRNA expressions may have therapeutic value for patients with AD.
Keywords: RNA silencing; Amyloid precursor protein; Tau; Voltage-dependent anion channel; Human neuroblastoma cell; Immunoblotting analysis;
BMP-6 inhibits cell proliferation by targeting microRNA-192 in breast cancer by Fen Hu; Xiangzhi Meng; Qi Tong; Lin Liang; Rong Xiang; Tianhui Zhu; Shuang Yang (2379-2390).
Although bone morphogenetic protein-6 (BMP-6) has been identified as a tumor suppressor associated with breast cancer differentiation and metastasis, the potential roles of BMP-6 in regulating cell cycle progression have not been fully examined. In the present study, we provide the novel finding that induction of BMP-6 in MDA-MB-231 breast cancer cells significantly inhibits cell proliferation by decreasing the number of cells in S phase of the cell cycle, resulting in inhibition of tumorigenesis in a nude mouse xenograft model. Further investigation indicated that BMP-6 up-regulates the expression of microRNA-192 (miR-192) in MDA-MB-231 cells. Elevated expression of miR-192 caused cell growth arrest, which is similar to the effect of BMP-6 induction. Importantly, depletion of endogenous miR-192 by miRNA inhibition significantly attenuated BMP-6-mediated repression of cell cycle progression. In breast cancer tissue, miR-192 expression is significantly down-regulated in tumor samples and positively correlates with the expression of BMP-6, demonstrating the inhibitory effect of BMP-6 on cell proliferation through miR-192 regulation. Additionally, using the RT2 Profiler PCR Array, retinoblastoma 1 (RB1) was identified as a direct target of the BMP-6/miR-192 pathway in regulating cell proliferation in breast cancer. In conclusion, we have identified an important role for BMP-6/miR-192 signaling in the regulation of cell cycle progression in breast cancer. Furthermore, BMP-6/miR-192 was expressed at low levels in breast cancer specimens, indicating that this pathway might represent a promising therapeutic target for breast cancer treatment.
Keywords: Bone morphogenetic protein-6; MicroRNA-192; Retinoblastoma 1; Cell proliferation; Breast cancer;
Functional heterogeneity of pulmonary surfactant protein-D in cystic fibrosis by Sailesh Kotecha; Iolo Doull; Philip Davies; Zofi McKenzie; Jens Madsen; Howard W. Clark; Eamon P. McGreal (2391-2400).
Pulmonary surfactant protein-D (SP-D) is a soluble collagenous C-type lectin with important anti-microbial and anti-inflammatory properties. Although it is subject to functionally relevant modification by common polymorphisms and unregulated inflammation, the functional status of SP-D in cystic fibrosis (CF) remains unclear. Given the importance of infection and inflammation in CF lung pathology we have undertaken the first systematic analysis of SP-D lectin activity in this population. By ELISA, we found that airway lavage fluid SP-D expression was greater in CF compared to control patients but was reduced in CF patients with infection and correlated negatively with markers of neutrophilic inflammation. In a functional assay, the percentage of SP-D capable of binding zymosan rarely exceeded 60% in CF or control patients and similarly restricted binding activity was observed towards maltose–agarose. SP-D lectin activity also correlated negatively with infection and neutrophilic inflammation but there was little evidence of major proteolytic degradation amongst the non-bound material. SP-D which failed to bind zymosan exhibited features of lower oligomeric form compared to bound material when tested by native gel electrophoresis. Furthermore, when separated by gel chromatography, high and low oligomeric populations of SP-D were observed in CF lavage fluid but only high oligomeric forms exhibited substantial lectin activity towards yeast derived mannan. Our data demonstrate that oligomeric heterogeneity underlies functional diversity amongst SP-D in health and disease and that dynamic regulation of oligomerisation is an important feature of SP-D biology.
Keywords: Cystic fibrosis; Innate immunity; Lung; Surfactant protein-D; Lectin; Inflammation;
Towards modern genetics, diagnostics and therapeutics of heart failure in a new era by Leon J. De Windt; Pieter A. Doevendans (2401-2402).
Regulation of expression of atrial and brain natriuretic peptide, biomarkers for heart development and disease by Irina A. Sergeeva; Vincent M. Christoffels (2403-2413).
The mammalian heart expresses two closely related natriuretic peptide (NP) hormones, atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). The excretion of the NPs and the expression of their genes strongly respond to a variety of cardiovascular disorders. NPs act to increase natriuresis and decrease vascular resistance, thereby decreasing blood volume, systemic blood pressure and afterload. Plasma levels of BNP are used as diagnostic and prognostic markers for hypertrophy and heart failure (HF), and both ANF and BNP are widely used in biomedical research to assess the hypertrophic response in cell culture or the development of HF related diseases in animal models. Moreover, ANF and BNP are used as specific markers for the differentiating working myocardium in the developing heart, and the ANF promoter serves as platform to investigate gene regulatory networks during heart development and disease. However, despite decades of research, the mechanisms regulating the NP genes during development and disease are not well understood. Here we review current knowledge on the regulation of expression of the genes for ANF and BNP and their role as biomarkers, and give future directions to identify the in vivo regulatory mechanisms. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.
Keywords: Natriuretic peptide; ANF; BNP; Gene regulation; Development; Heart failure;
Regulation of fetal gene expression in heart failure by Ellen Dirkx; Paula A. da Costa Martins; Leon J. De Windt (2414-2424).
During the processes leading to adverse cardiac remodeling and heart failure, cardiomyocytes react to neurohumoral stimuli and biomechanical stress by activating pathways that induce pathological hypertrophy. The gene expression patterns and molecular changes observed during cardiac hypertrophic remodeling bare resemblance to those observed during fetal cardiac development. The re-activation of fetal genes in the adult failing heart is a complex biological process that involves transcriptional, posttranscriptional and epigenetic regulation of the cardiac genome. In this review, the mechanistic actions of transcription factors, microRNAs and chromatin remodeling processes in regulating fetal gene expression in heart failure are discussed.
Keywords: Gene regulation; Transcription factors; Epigenetics;
Alterations in ryanodine receptors and related proteins in heart failure by Sameer Ather; Jonathan L. Respress; Na Li; Xander H.T. Wehrens (2425-2431).
Sarcoplasmic reticulum (SR) Ca2 + release plays an essential role in mediating cardiac myocyte contraction. Depolarization of the plasma membrane results in influx of Ca2 + through l-type Ca2 + channels (LTCCs) that in turn triggers efflux of Ca2 + from the SR through ryanodine receptor type-2 channels (RyR2). This process known as Ca2 +-induced Ca2 +release (CICR) occurs within the dyadic region, where the adjacent transverse (T)-tubules and SR membranes allow RyR2 clusters to release SR Ca2 + following Ca2 + influx through adjacent LTCCs. SR Ca2 + released during systole binds to troponin-C and initiates actin–myosin cross-bridging, leading to muscle contraction. During diastole, the cytosolic Ca2 + concentration is restored by the resequestration of Ca2 + into the SR by SR/ER Ca2 +-ATPase (SERCA2a) and by the extrusion of Ca2 + via the Na+/Ca2 +-exchanger (NCX1). This whole process, entitled excitation–contraction (EC) coupling, is highly coordinated and determines the force of contraction, providing a link between the electrical and mechanical activities of cardiac muscle. In response to heart failure (HF), the heart undergoes maladaptive changes that result in depressed intracellular Ca2 + cycling and decreased SR Ca2 + concentrations. As a result, the amplitude of CICR is reduced resulting in less force production during EC coupling. In this review, we discuss the specific proteins that alter the regulation of Ca2 + during HF. In particular, we will focus on defects in RyR2-mediated SR Ca2 + release. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.
Keywords: Heart failure; Calcium; Ryanodine receptor; Sarcoplasmic reticulum; Contractility;
Electrophysiological changes in heart failure and their implications for arrhythmogenesis by R. Coronel; Ronald Wilders; Arie O. Verkerk; Rob F. Wiegerinck; David Benoist; Olivier Bernus (2432-2441).
Heart failure is the final common pathway of various cardiac pathologies and is associated with sudden cardiac death, mostly caused by ventricular arrhythmias. In this paper we briefly review the electrophysiological remodeling and the alterations in intracellular calcium handling, and the resulting arrhythmogenic mechanisms associated with heart failure. Intercellular uncoupling and fibrosis are identified as a major arrhythmogenic factors. Diet and ventricular wall stretch are discussed as modulating factors. Finally, emphasis is placed on the hitherto poorly studied aspects of right ventricular failure. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.Display Omitted
Keywords: Heart failure; Arrhythmia mechanism; Intercellular coupling; Diet; Stretch; Pulmonary hypertension;
Biomarkers and diagnostics in heart failure by Hanna K. Gaggin; James L. Januzzi (2442-2450).
Heart failure (HF) biomarkers have dramatically impacted the way HF patients are evaluated and managed. B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are the gold standard biomarkers in determining the diagnosis and prognosis of HF, and studies on natriuretic peptide-guided HF management look promising. An array of additional biomarkers has emerged, each reflecting different pathophysiological processes in the development and progression of HF: myocardial insult, inflammation and remodeling. Novel biomarkers, such as mid-regional pro atrial natriuretic peptide (MR-proANP), mid-regional pro adrenomedullin (MR-proADM), highly sensitive troponins, soluble ST2 (sST2), growth differentiation factor (GDF)-15 and Galectin-3, show potential in determining prognosis beyond the established natriuretic peptides, but their role in the clinical care of the patient is still partially defined and more studies are needed. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.► BNP and NT-proBNP are gold standard HF biomarkers in diagnosis and prognosis of HF. ► BNP and NT-proBNP-guided HF management look promising. ► Novel biomarkers include MR-proANP, MR-proADM, troponins, sST2, GDF-15, galectin-3. ► Novel biomarkers show promise in HF prognosis beyond BNP and NT-proBNP. ► The role of novel biomarkers in clinical care is still only partially defined.
Keywords: Heart failure; Biomarker; Diagnosis; Prognosis; Management;
Genetics of heart failure by Luís R. Lopes; Perry M. Elliott (2451-2461).
Heart failure (HF) occurs when the cardiac output, no longer compensated by endogenous mechanisms, fails to meet the metabolic demands of the body. In most populations, the prevalence of heart failure continues to rise, constituting a major public health burden, especially in developed countries. There is some evidence that the risk of HF in the general population depends on genetic predisposition, necessarily characterised by a very complex architecture. In a small, but probably underestimated proportion, HF is caused by Mendelian inherited forms of myocardial disease. The genetic background of these genetic conditions is a matter of intensive research that is already shedding light onto the genetics of common sporadic forms of HF. In this review, we briefly review the insights provided by candidate gene and genome-wide association approaches in common HF and then describe the main genetic causes of inherited heart muscle disease. Finally we present the current challenges and future research needs for both forms of HF. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.► The risk of heart failure (HF) in the population depends on genetic predisposition. ► The genetic background of HF is characterised by a very complex architecture. ► We review candidate gene and genome-wide association approaches in common HF. ► We describe the genetic causes of inherited heart muscle disease. ► We further present the challenges and future research needs for both forms of HF.
Keywords: Heart failure; Genetics; Candidate gene study; GWAS; High-throughput sequencing; Cardiomyopathy;