BBA - Molecular Basis of Disease (v.1812, #11)

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease affecting thousands of people in the world and effective treatment is still not available. Over two decades of intense research using AD postmortem brains, transgenic mouse and cell models of amyloid precursor protein and tau revealed that amyloid beta (Aβ) and hyperphosphorylated tau are synergistically involved in triggering disease progression. Accumulating evidence also revealed that aging and amyloid beta-induced oxidative DNA damage and mitochondrial dysfunction initiate and contributes to the development and progression of the disease. The purpose of this article is to summarize the latest progress in aging and AD, with a special emphasis on the mitochondria, oxidative DNA damage including methods of its measurement. It also discusses the therapeutic approaches against oxidative DNA damage and treatment strategies in AD.► This article summarizes recent developments of aging and amyloid beta-induced oxidative DNA damage in Alzheimer's disease. ► Discussed the factors that may be responsible for the development of Alzheimer's disease. ► Methods of detection of oxidative DNA damage were discussed. ► Discussed the therapeutic approaches of oxidative DNA damage.
Keywords: Amyloid-β; Oxidative stress; Antioxidant; Telomere; p53; DNA repair;

1H NMR based metabolomics of CSF and blood serum: A metabolic profile for a transgenic rat model of Huntington disease by Kim A. Verwaest; Trung N. Vu; Kris Laukens; Laura E. Clemens; Huu P. Nguyen; Bjorn Van Gasse; José C. Martins; Annemie Van Der Linden; Roger Dommisse (1371-1379).
Huntington disease (HD) is a hereditary brain disease. Although the causative gene has been found, the exact mechanisms of the pathogenesis are still unknown. Recent investigations point to metabolic and energetic dysfunctions in HD neurons.Both univariate and multivariate analyses were used to compare proton nuclear magnetic resonance spectra of serum and cerebrospinal fluid (CSF) taken from presymptomatic HD transgenic rats and their wild-type littermates. N-acetylaspartate (NAA), was found to be significantly decreased in the serum of HD rats compared to wild-type littermates. Moreover, in the serum their levels of glutamine, succinic acid, glucose and lactate are significantly increased as well. An increased concentration of lactate and glucose is also found in CSF. There is a 1:1 stoichiometry coupling glucose utilization and glutamate cycling. The observed increase in the glutamine concentration, which indicates a shutdown in the neuronal-glial glutamate-glutamine cycling, results therefore in an increased glucose concentration. The elevated succinic acid concentration might be due to an inhibition of succinate dehydrogenase, an enzyme linked to the mitochondrial respiratory chain and TCA cycle. Moreover, reduced levels of NAA may reflect an impairment of mitochondrial energy production. In addition, the observed difference in lactate supports a deficiency of oxidative energy metabolism in rats transgenic for HD as well.The observed metabolic alterations seem to be more profound in serum than in CSF in presymptomatic rats. All findings suggest that even in presymptomatic rats, a defect in energy metabolism is already apparent. These results support the hypothesis of mitochondrial energy dysfunction in HD. 1H NMR based metabolomics study of rats transgenic for Huntington disease. ► Both in serum and CSF metabolic differences are observed in presymptomatic rats. ► The metabolic disturbance is more profound in serum than in CSF. ► All findings indicate a defect in energy metabolism in the transgenic rats.
Keywords: Huntington disease; CSF; Serum; Metabolomics; Transgenic rat; 1H NMR spectroscopy;

A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: Novel avenues for biochemical and therapeutic studies by M. Kranendijk; G.S. Salomons; K.M. Gibson; E. Van Schaftingen; C. Jakobs; E.A. Struys (1380-1384).
The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg140 to Gln140 or Gly140 (IDH2wt/R140Q, IDH2wt/R140G) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1R132H and IDH2R172K mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2wt/R140Q gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2wt/R140Q enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-2H4]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2wt/R140Q enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4 nmol h−1  mg protein−1 vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2wt/R140Q activity was obtained with oxaloacetate, which competitively inhibited IDH2wt/R140Q activity. Lymphoblast IDH2wt/R140Q showed long-term cell culture stability without loss of the heterozygous IDH2wt/R140Q mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.► IDH2R140Q gain-of-function enzyme assay developed in lymphoblasts with UPLC-MS/MS. ► 8 times increased IDH2R140Q reaction rate is found in D-2-HGA type II vs. controls. ► Intracellular D-2-HG levels are increased 140 times in D-2-HGA type II lymphoblasts. ► IDH2R140Q reaction rate was inhibited with oxaloacetate reducing D-2-HG production. ► Lymphoblast model creates novel avenues for biochemical and therapeutic studies.
Keywords: D-2-hydroxyglutaric aciduria; 2-hydroxyglutarate; Isocitrate dehydrogenase 2; IDH2 R140Q gain-of-function; Enzyme assay; Lymphoblast model;

F508del-CFTR increases intracellular Ca2+ signaling that causes enhanced calcium-dependent Cl conductance in cystic fibrosis by Joana Raquel Martins; Patthara Kongsuphol; Eva Sammels; Shehrazade Dahimène; Fadi AlDehni; Luka Alexander Clarke; Rainer Schreiber; Humbert de Smedt; Margarida D. Amaral; Karl Kunzelmann (1385-1392).
In many cells, increase in intracellular calcium ([Ca2+]i) activates a Ca2+-dependent chloride (Cl) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca2+]i increase is Cl dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca2+. This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca2+]i. Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP3 (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca2+ signaling.► Enhanced Ca2+ activated Cl currents in CF is not caused by enhanced expression of TMEM16A and bestrophin 1. ► Calcium signaling was enhanced after induction of expression of F508del-CFTR. ► Receptor-mediated [Ca2+]i increase is enhanced by F508del-CFTR which serves as counter-ion channel for Ca2+ in the ER. ► The double mutant F508del/G551D-CFTR remains trapped in the ER but has no effects on [Ca2+]i.► Ca2+ signaling and Ca2+ dependent Cl conductance are enhanced in CF cells independent of infection.
Keywords: TMEM16A; Bestrophin 1; Ca2+ activated Cl current; CF inflammation; Purinergic receptor; Cl channel;

Inherited glucose-6-phosphate isomerase (GPI) deficiency is the second most frequent glycolytic erythroenzymopathy in humans. Patients present with non-spherocytic anemia of variable severity and with neuromuscular dysfunction. We previously described Chinese hamster (CHO) cell lines with mutations in GPI and loss of GPI activity. This resulted in a temperature sensitivity and severe reduction in the synthesis of glycerolipids due to a reduction in phosphatidate phosphatase (PAP). In the current article we attempt to describe the nature of this pleiotropic effect. We cloned and sequenced the CHO lipin 1 cDNA, a gene that codes for PAP activity. Overexpression of lipin 1 in the GPI-deficient cell line, GroD1 resulted in increased PAP activity, however it failed to restore glycerolipid biosynthesis. Fluorescence microscopy showed a failure of GPI-deficient cells to localize lipin 1α to the nucleus. We also found that glucose-6-phosphate levels in GroD1 cells were 10-fold over normal. Lowering glucose levels in the growth medium partially restored glycerolipid biosynthesis and nuclear localization of lipin 1α. Western blot analysis of the elements within the mTOR pathway, which influences lipin 1 activity, was consistent with an abnormal activation of this system. Combined, these data suggest that GPI deficiency results in an accumulation of glucose-6-phosphate, and possibly other glucose-derived metabolites, leading to activation of mTOR and sequestration of lipin 1 to the cytosol, preventing its proper functioning. These results shed light on the mechanism underlying the pathologies associated with inherited GPI deficiency and the variability in the severity of the symptoms observed in these patients.► Lipin 1α mislocalization is due to a mutation in the GPI gene. ► Demonstrated glucose sensitivity in a GPI-deficient cell line. ► Altered lipid biosynthesis as a pathological factor in inherited GPI deficiency. ► First sequencing of lipin 1 cDNA from hamster.
Keywords: Glucose-6-phosphate isomerase deficiency; Mutant; Phospholipid biosynthesis; Lipin 1; Lipodystrophy; Non-spherocytic anemia;

Downregulation of the Wnt antagonist Dkk2 links the loss of Sept4 and myofibroblastic transformation of hepatic stellate cells by Atsuko Yanagida; Keiko Iwaisako; Etsuro Hatano; Kojiro Taura; Fumiaki Sato; Masato Narita; Hiromitsu Nagata; Hiroyuki Asechi; Shinji Uemoto; Makoto Kinoshita (1403-1411).
Sept4, a subunit of the septin cytoskeleton specifically expressed in quiescent hepatic stellate cells (HSCs), is downregulated through transdifferentiation to fibrogenic and contractile myofibroblastic cells. Since Sept4 −/− mice are prone to liver fibrosis, we aimed to identify the unknown molecular network underlying liver fibrosis by probing the association between loss of Sept4 and accelerated transdifferentiation of HSCs.We compared the transcriptomes of Sept4 +/+ and Sept4 −/− HSCs undergoing transdifferentiation by DNA microarray and quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Because Dickkopf2 (Dkk2) gene expression is reduced in Sept4 −/− HSCs, we tested whether supplementing Dkk2 could suppress myofibroblastic transformation of Sept4 −/− HSCs. We tested the involvement of the canonical Wnt pathway in this process by using a lymphoid enhancer-binding factor/transcription factor-luciferase reporter assay.We observed consistent upregulation of Dkk2 in primary cultured HSCs and in a carbon tetrachloride liver fibrosis in mice, which was decreased in the absence of Sept4. Supplementation with Dkk2 suppressed the induction of pro-fibrotic genes (α-smooth muscle actin and 2 collagen genes) and induced an anti-fibrotic gene (Smad7) in Sept4 −/− HSCs. In human liver specimens with inflammation and fibrosis, Dkk2 immunoreactivity appeared to be positively correlated with the degree of fibrotic changes.Pro-fibrotic transformation of HSCs through the loss of Sept4 is, in part, due to reduced expression of Dkk2 and its homologues, and the resulting disinhibition of the canonical Wnt pathway.► Mice without a hepatic stellate cell(HSC)-specific gene Sept4 develop liver fibrosis. ► Unbiased screening of Sept4-null HSCs revealed downregulation of the Dickkopf genes. ► Dickkopfs are the Wnt inhibitors implicated in liver fibrosis in mice and humans. ► Supplementing HSCs with Dickkopf2 suppressed the upregulation of pro-fibrotic genes. ► Disinhibited Wnt signals account for the pro-fibrotic phenotype of Sept4-null mice.
Keywords: Septin; Hepatic stellate cell; Canonical Wnt pathway; Dkk (Dickkopf); Myofibroblastic transformation; Liver fibrosis;

Increased efflux of oxidized glutathione (GSSG) causes glutathione depletion and potentially diminishes antioxidant defense in sickle erythrocytes by Erfan Nur; Mirjam Verwijs; Dirk R. de Waart; John-John B. Schnog; Hans-Martin Otten; Dees P. Brandjes; Bart J. Biemond; Ronald P.J. Oude Elferink (1412-1417).
Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 μmol/l vs. 40.6 ± 6.9 μmol/l and 25.8 ± 2.7 μmol/l vs. 13.6 ± 1.7 μmol/l respectively, P  < 0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 μmol/l vs. 163 ± 13.6 μmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 μmol/l to 111 ± 11.8 μmol/l, P  < 0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress.► Sickle cell disease characterized by increased oxidative stress and poor antioxidant capacity. ► Decreased intracellular GSH levels in sickle erythrocytes. ► GSSG efflux increased in sickle erythrocytes, causing net loss of GSH. ► Increased GSSG efflux mediated by MRP1 transporters.
Keywords: Sickle cell disease; Oxidative stress; Glutathione; GSH; GSSG;

Myelin genes are downregulated in canine fucosidosis by Jessica L. Fletcher; Gauthami S. Kondagari; Amanda L. Wright; Peter C. Thomson; Peter Williamson; Rosanne M. Taylor (1418-1426).
The processes regulating the complex neurodegenerative cascade of vacuolation, neuroinflammation, neuronal loss and myelin deficits in fucosidosis, a neurological lysosomal storage disorder, remain unclear. To elucidate these processes the gene expression profile of the cerebral cortex from untreated and intrathecal enzyme replacement therapy treated fucosidosis pups and age-matched unaffected controls were examined. Neuroinflammation and cell death processes were identified to have a major role in fucosidosis pathophysiology with 37% of differentially expressed (DE) genes involved in these processes. Critical, specific, early decreases in expression levels of key genes in myelin assembly were identified by gene expression profiling, including myelin-associated glycoprotein (MAG), myelin and lymphocyte protein (MAL), and oligodendrocyte myelin paranodal and inner loop protein (OPALIN). These gene expression changes may be indicative of early neuronal loss causing reduced electrical impulses required for oligodendrocyte maturation.► Oligodendrocyte and myelin genes are downregulated in early canine fucosidosis. ► Neuroinflammation and apoptosis are key disease processes. ► Low dose, monthly enzyme replacement therapy does not significantly affect global gene expression.
Keywords: Lysosomal storage disease; Fucosidosis; Hypomyelination; Microarray; Canine disease model;

Oxidative stress is a feature of cholestatic syndrome and induces multidrug resistance-associated protein 2 (Mrp2) internalization from the canalicular membrane surface. We have previously shown that the activation of a novel protein kinase C (nPKC) by oxidative stress regulates Mrp2 internalization. The internalized Mrp2 was recycled to the canalicular surface in a protein kinase A (PKA)-dependent manner after intracellular glutathione (GSH) levels were replenished. However, the putative phosphorylation targets of these protein kinases involved in reversible Mrp2 trafficking remain unclear. In this study, we investigated the effect of changing the intrahepatic redox status on the C-terminal phosphorylation status of radixin (p-radixin), which links Mrp2 to F-actin, and the interaction of p-radixin with Mrp2 in rat hepatocytes. We detected a significant decrease in the amount of p-radixin that co-immunoprecipitated with Mrp2 after tertiary-butylhydroperoxide (t-BHP) treatment. After treatment with GSH-ethylester (GSH-EE), the phosphorylation level became the same as that of the control. A PKC and protein phosphatase (PP)-1/2A inhibitor, but not a PP-2A selective inhibitor, prevented the t-BHP-induced decrease of p-radixin and subsequent canalicular Mrp2 localization. In contrast, a PKA inhibitor affected the recovery process facilitated by GSH-EE treatment. In conclusion, the interaction of p-radixin with Mrp2 was decreased by the activation of PKC and PP-1 under oxidative stress conditions which subsequently led to Mrp2 internalization, whereas the interaction of p-radixin and Mrp2 was increased by the activation of PKA during recovery from oxidative stress.► The interhepatic GSH regulates the balance of activation of PKA and PKC. ► Activated protein kinases determine the phosphorylation status of radixin. ► The phosphorylation status of radixin regulates the interaction with Mrp2. ► The interaction of radixin with Mrp2 determines the canalicular Mrp2 localization.
Keywords: Mrp2; Radixin; Cholestasis; Oxidative stress; Internalization;

Natural phenylalanine hydroxylase variants that confer a mild phenotype affect the enzyme's conformational stability and oligomerization equilibrium by Monica Cerreto; Paola Cavaliere; Carla Carluccio; Felice Amato; Adriana Zagari; Aurora Daniele; Francesco Salvatore (1435-1445).
Hyperphenylalaninemias are genetic diseases prevalently caused by mutations in the phenylalanine hydroxylase (PAH) gene. The wild-type PAH enzyme is a homotetramer regulated by its substrate, cofactor and phosphorylation. We reproduced a full-length wild-type protein and seven natural full-length PAH variants, p.I65M, p.N223Y, p.R297L, p.F382L, p.K398N, p.A403V, and p.Q419R, and analyzed their biochemical and biophysical behavior. All mutants exhibited reduced enzymatic activity, namely from 38% to 69% of wild-type activity. Biophysical characterization was performed by size-exclusion chromatography, light scattering and circular dichroism. In the purified wild-type PAH, we identified the monomer in equilibrium with the dimer and tetramer. In most mutants, the equilibrium shifted toward the dimer and most tended to form aggregates. All PAH variants displayed different biophysical behaviors due to loss of secondary structure and thermal destabilization. Specifically, p.F382L was highly unstable at physiological temperature. Moreover, using confocal microscopy with the number and brightness technique, we studied the effect of BH4 addition directly in living human cells expressing wild-type PAH or p.A403V, a mild mutant associated with BH4 responsiveness in vivo. Our results demonstrate that BH4 addition promotes re-establishment of the oligomerization equilibrium, thus indicating that the dimer-to-tetramer shift in pA403V plays a key role in BH4 responsiveness. In conclusion, we show that the oligomerization process and conformational stability are altered by mutations that could affect the physiological behavior of the enzyme. This endorses the hypothesis that oligomerization and folding defects of PAH variants are the most common causes of HPAs, particularly as regards mild human phenotypes.► Physico-chemical features of the wild-type and of seven PAH mutants are described. ► PAH natural mutants show thermolability and folding defects. ► The mutant protein equilibrium is shifted versus the dimer and higher aggregates. ► p.A403V BH4 responsiveness is shown in vivo with the N&B technique.
Keywords: BH4 responsiveness; Number and brightness; PAH conformational stability; PAH oligomerization equilibrium; Hyperphenylalaninemia; Phenylalanine hydroxylase;

Influence of aging and hemorrhage injury on Sirt1 expression: Possible role of myc-Sirt1 regulation in mitochondrial function by Bixi Jian; Shaolong Yang; Dongquan Chen; Irshad Chaudry; Raghavan Raju (1446-1451).
Trauma–hemorrhage (T–H) causes hypoxia and organ dysfunction. Mitochondrial dysfunction is a major factor for cellular injury due to T–H. Aging also has been known to cause progressive mitochondrial dysfunction. In order to study the effect of aging on T–H-induced mitochondrial dysfunction, we recently developed a rodent mitochondrial genechip with probesets representing mitochondrial and nuclear genes contributing to mitochondrial structure and function. Using this chip we recently identified signature mitochondrial genes altered following T–H in 6 and 22 month old rats; augmented expression of the transcription factor c-myc was the most pronounced. Based on reports of c-myc-IL6 collaboration and c-myc-Sirt1 negative regulation, we further investigated the expression of these regulatory factors with respect to aging and injury. Rats of ages 6 and 22 months were subjected to T–H or sham operation and left ventricular tissues were tested for cytosolic cytochrome c, mtDNA content, Sirt1 and mitochondrial biogenesis factors Foxo1, Ppara and Nrf-1. We observed increased cardiac cytosolic cytochrome c (sham vs T–H, p  < 0.03), decreased mitochondrial DNA content (sham vs T–H, p  < 0.05), and decreased Sirt1 expression (sham vs TH, p  < 0.05) following T–H and with progressing age. Additionally, expression of mitochondrial biogenesis regulating transcription factors Foxo1 and Nrf-1 was also decreased with T–H and aging. Based upon these observations we conclude that Sirt1 expression is negatively modulated by T–H causing downregulation of mitochondrial biogenesis. Thus, induction of Sirt1 is likely to produce salutary effects following T–H induced injury and hence, Sirt1 may be a potential molecular target for translational research in injury resolution.► Severe hemorrhage causes cardiac mitochondrial dysfunction. ► Aging exacerbates mitochondrial functional alteration due to hemorrhage. ► We observed increased release of cytochrome c from mitochondria to cytosol with aging and injury. ► We found a decrease in Sirt1 expression following hemorrhage. ► Coupled with our previous observation that c-Myc is upregulated following hemorrhage, our hypothesis is that c-Myc-Sirt1 regulation is important in mitochondrial function following hemorrhage injury.
Keywords: Hemorrhage; Trauma; Aging; Blood loss; Trauma–hemorrhage; Cardiac function; Senescence;

Identification and functional characterization of KCNQ1 mutations around the exon 7–intron 7 junction affecting the splicing process by Keiko Tsuji–Wakisaka; Masaharu Akao; Takahiro M. Ishii; Takashi Ashihara; Takeru Makiyama; Seiko Ohno; Futoshi Toyoda; Kenichi Dochi; Hiroshi Matsuura; Minoru Horie (1452-1459).
Background. KCNQ1 gene encodes the delayed rectifier K+ channel in cardiac muscle, and its mutations cause long QT syndrome type 1 (LQT1). Especially exercise-related cardiac events predominate in LQT1. We previously reported that a KCNQ1 splicing mutation displays LQT1 phenotypes. Methods and results. We identified novel mutation at the third base of intron 7 (IVS7 + 3A > G) in exercise-induced LQT1 patients. Minigene assay in COS7 cells and RT-PCR analysis of patients' lymphocytes demonstrated the presence of exon 7-deficient mRNA in IVS7 + 3A > G, as well as c.1032G > A, but not in c.1022C > T. Real-time RT-PCR demonstrated that both IVS7 + 3A > G and c.1032G > A carrier expressed significant amounts of exon-skipping mRNAs (18.8% and 44.8% of total KCNQ1 mRNA). Current recordings from Xenopus oocytes injected cRNA by simulating its ratios of exon skipping displayed a significant reduction in currents to 64.8 ± 4.5% for IVS7 + 3A > G and to 41.4 ± 9.5% for c.1032G > A carrier, respectively, compared to the condition without splicing error. Computer simulation incorporating these quantitative results revealed the pronounced QT prolongation under beta-adrenergic stimulation in IVS7 + 3A > G carrier model. Conclusion. Here we report a novel splicing mutation IVS7 + 3A > G, identified in a family with mild form LQT1 phenotypes, and examined functional outcome in comparison with three other variants around the exon 7–intron 7 junction. In addition to c.1032G > A mutation, IVS7 + 3A > G generates exon-skipping mRNAs, and thereby causing LQT1 phenotype. The severity of clinical phenotypes appeared to differ between the two splicing-related mutations and to result from the amount of resultant mRNAs and their functional consequences.► A novel KCNQ1 splicing mutation IVS7 + 3A > G generates exon-skipping mRNAs. ► In addition to c.1032G > A mutation, novel IVS7 + 3A > G mutation causes LQT1 phenotype. ► The severity of clinical phenotypes appeared to differ between the two mutations. ► The amount of exon-skipping mRNAs may determine the clinical severity of the disease.
Keywords: Long QT syndrome; Ion channel; Splicing mutation;

Differential effects of insulin on peripheral diabetes-related changes in mitochondrial bioenergetics: Involvement of advanced glycosylated end products by Aline Pertile Remor; Filipe José de Matos; Karina Ghisoni; Thiago Lenoir da Silva; Greici Eidt; Marília Búrigo; Andreza Fabro de Bem; Paulo César Lock Silveira; Andrés de León; Maria Cecilia Sanchez; Alexandre Hohl; Viviane Glaser; Carlos-Alberto Gonçalves; André Quincozes-Santos; Rafael Borba Rosa; Alexandra Latini (1460-1471).
Large scale clinical trials have demonstrated that an intensive antihyperglycemic treatment in diabetes mellitus (DM) in individuals reduces the incidence of micro- and macrovascular complications, e.g. nephropathy, retinopathy, DM-accelerated atherosclerosis, myocardial infarction, or limb amputations. Here, we investigated the effect of short- and long-term insulin administration on mitochondrial function in peripheral tissues of streptozotocin (STZ)-induced hyperglycemic rats. In addition, the in vitro effect of methylglyoxal (MG), advanced glycation end products (AGEs) and human diabetic plasma on mitochondrial activity was investigated in skeletal muscle and liver mitochondria and in rat skin primary fibroblasts. Hyperglycemic STZ rats showed tissue-specific patterns of energy deficiency, evidenced by reduced activities of complexes I, II and/or IV after 30 days of hyperglycemia in heart, skeletal muscle and liver; moreover, cardiac tissue was found to be the most sensitive to the diabetic condition, since energy metabolism was impaired after 10 days of the hyperglycemia. Insulin-induced tight glycemic control was effective in protecting against the hyperglycemia-induced inhibition of mitochondrial enzyme activities. Furthermore, the long-term hormone replacement (30 days) also increased these activities in kidney from STZ-treated animals, where the hyperglycemic state did not modify the electron transport activity. Results from in vitro experiments indicate that mitochondrial impairment could result from oxidative stress-induced accumulation of MG and/or AGEs. Further investigations demonstrated that human plasma AGE accumulation elicits reduced mitochondrial function in skin fibroblast. These data suggest that persistent hyperglycemia results in tissue-specific patterns of energy deficiency and that early and continuous insulin therapy is necessary to maintain proper mitochondrial metabolism.► Persistent hyperglycemia results in tissue-specific patterns of energy deficiency. ► Insulin administration had protective effects in peripheral tissues in STZ rats. ► Tight glucose control is essential to avoid mitochondrial impairment in STZ rats. ► Human plasma accumulating AGEs impairs mitochondrial activity in cultured fibroblasts. ► Energy deficit was possibly caused by AGE inducing mitochondrial oxidative stress.
Keywords: Hyperglycemia; Insulin; Mitochondrial oxidative stress; Cardioprotection; Streptozotocin;

Retinitis pigmentosa, causing progressive blindness, is genetically heterogeneous. RP10, due to a defect in inosine monophosphate dehydrogenase 1 (IMPDH1), shows autosomal dominant inheritance. Recombinantly expressed clinical mutants show unaltered kinetic behaviour. It is unclear why reportedly impaired DNA binding is important and how it would explain negative dominance. An alternative view relates to the mutant proteins' tendency to aggregate. Regarding negative dominance, a key question is whether the defective protein can subvert the function of its normal counterpart in the same cell. Potentially, the homotetrameric structure of IMPDH1 might offer a vehicle for such an effect. We have established a reliable protocol for reproducible refolding of recombinantly expressed IMPDH1 in vitro. Clinical mutants R224P and D226N both show impaired folding. For equimolar mixtures of normal and mutant enzymes, independent refolding would predict activity regain midway between pure mutant and pure normal. Under various conditions regain is close to the mutant figure, suggesting that, in hybrid tetramers, mutant subunits impose their faulty conformation on normal partners. The observed molecular recruitment is a negative counterpart of the intra-allelic complementation, also mediated via oligomeric structure and postulated many years ago by Fincham. These findings appear potentially to account for the negative dominant inheritance. This interpretation must be provisional at present, as the predominant transcript in retina is an alternatively spliced version not fully identical to that used in our study. The results nevertheless have a general significance in pointing to a mechanism for negative dominance that could be widespread.► IMPDH1 can be successfully refolded and purified from inclusion bodies. ► Soluble IMPDH1 can be unfolded with guanidinium chloride and refolded in high yield. ► RP10 mutants R224P, D226N of IMPDH1 refold poorly and impair folding of normal IMPDH1. ► CD studies confirm faulty structure and thermal instability in IMPDH1 mutant D226N. ► Molecular recruitment by faulty subunits explains negative dominance in RP10.
Keywords: Retinitis pigmentosa RP10; Inosine monophosphate dehydrogenase 1 (IMPDH1); Dominant negative mutation; Protein folding; Subunit interaction; Protein aggregation;

Calorie restriction and resveratrol in cardiovascular health and disease by Vernon W. Dolinsky; Jason R.B. Dyck (1477-1489).
Calorie restriction is one of the most effective nutritional interventions that reproducibly protects against obesity, diabetes and cardiovascular disease. Recent evidence suggests that even when implemented over a short period, calorie restriction is a safe and effective treatment for cardiovascular disease. Herein, we review the effects of calorie restriction on the cardiovascular system as well as the biological effects of resveratrol, the most widely studied molecule that appears to mimic calorie restriction. An overview of microarray data reveals that the myocardial transcriptional effects of calorie restriction overlap with the transcriptional responses to resveratrol treatment. In addition, calorie restriction and resveratrol modulate similar pathways to improve mitochondrial function, reduce oxidative stress and increase nitric oxide production that are involved in atherosclerosis prevention, blood pressure reduction, attenuation of left-ventricular hypertrophy, resistance to myocardial ischemic injury and heart failure prevention. We also review the data that suggest that the effects of calorie restriction and resveratrol on the cardiovascular system may involve signaling through the silent information regulator of transcription (SIRT), Akt and the AMP-activated protein kinase (AMPK) pathways. While accumulating data demonstrate the health benefits of calorie restriction and resveratrol in experimental animal models, whether these interventions translate to patients with cardiovascular disease remains to be determined.► Calorie restriction (CR) prevents cardiovascular disease (CVD) in many experimental animal models. ► Treatment of experimental animals with resveratrol (Resv) reproduces many of the benefits of CR for CVD. ► CR and Resv reduce CVD via increased insulin sensitivity and nitric oxide production and reduced oxidative stress and inflammation. ► At a molecular level, CR and Resv share common targets that mediate their biological activities.
Keywords: Cardiovascular disease; Calorie restriction; Resveratrol; Sirtuin; AMP-activated protein kinase; Nitric oxide;

Molecular machinery of macroautophagy and its deregulation in diseases by Alan S.L. Wong; Zelda H. Cheung; Nancy Y. Ip (1490-1497).
Macroautophagy maintains cellular homeostasis through targeting cytoplasmic contents and organelles into autophagosomes for degradation. This process begins with the assembly of protein complexes on isolation membrane to initiate the formation of autophagosome, followed by its nucleation, elongation and maturation. Fusion of autophagosomes with lysosomes then leads to degradation of the cargo. In the past decade, significant advances have been made on the identification of molecular players that are implicated in various stages of macroautophagy. Post-translational modifications of macroautophagy regulators have also been demonstrated to be critical for the selective targeting of cytoplasmic contents into autophagosomes. In addition, recent demonstration of distinct macroautophagy regulators has led to the identification of different subtypes of macroautophagy. Since deregulation of macroautophagy is implicated in diseases including neurodegenerative disorders, cancers and inflammatory disorders, understanding the molecular machinery of macroautophagy is crucial for elucidating the mechanisms by which macroautophagy is deregulated in these diseases, thereby revealing new potential therapeutic targets and strategies. Here we summarize current knowledge on the regulation of mammalian macroautophagy machineries and their disease-associated deregulation.► Macroautophagy begins with the initiation of autophagosome formation. ► Autophagosome formation involves its nucleation, elongation and maturation. ► Macroautophagy regulators control selective degradation of cytoplasmic contents. ► Macroautophagy deregulation implicated in cancers, neurodegenerative and inflammatory diseases. ► Macroautophagy is deregulated via distinct mechanisms in different diseases.
Keywords: Autophagy; Autophagosome; Cdk5; Endophilin B1; Post-translational modification; Neurodegenerative disorder;

PP2A targeting by viral proteins: A widespread biological strategy from DNA/RNA tumor viruses to HIV-1 by Julien Guergnon; Angélique N. Godet; Amandine Galioot; Pierre Barthélémy Falanga; Jean-Hervé Colle; Xavier Cayla; Alphonse Garcia (1498-1507).
Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as constitutive housekeeping enzymes, it is now well established that PP2A proteins represent a family of highly and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A targeting for research in human diseases, and in further therapeutic developments, is also discussed.► The family of PP2A holoenzymes are specific targets for multiple viruses. ► Viruses use PP2A to specifically subvert key survival pathways of their hosts. ► Proteins encoded by DNA/RNA tumor viruses bind PP2A to activate oncogenic pathways. ► PP2A dysfunction is associated to cancer and neurodegeneration. ► The study of PP2A dysfunction is a powerful approach for human therapy.
Keywords: Viruses; Cancer; Neurodegeneration; PP2A holoenzymes; PP2A and human therapy;

Chemical characterization of pro-inflammatory amyloid-beta peptides in human atherosclerotic lesions and platelets by Tyler A. Kokjohn; Gregory D. Van Vickle; Chera L. Maarouf; Walter M. Kalback; Jesse M. Hunter; Ian D. Daugs; Dean C. Luehrs; John Lopez; Daniel Brune; Lucia I. Sue; Thomas G. Beach; Eduardo M. Castaño; Alex E. Roher (1508-1514).
Amyloid-β (Aβ) peptides are intimately involved in the inflammatory pathology of atherosclerotic vascular disease (AVD) and Alzheimer's disease (AD). Although substantial amounts of these peptides are produced in the periphery, their role and significance to vascular disease outside the brain requires further investigation. Amyloid-β peptides present in the walls of human aorta atherosclerotic lesions as well as activated and non-activated human platelets were isolated using sequential size-exclusion columns and HPLC reverse-phase methods. The Aβ peptide isolates were quantified by ELISA and structurally analyzed using MALDI-TOF mass spectrometry procedures. Our experiments revealed that both aorta and platelets contained Aβ peptides, predominately Aβ40. The source of the Aβ pool in aortic atherosclerosis lesions is probably the activated platelets and/or vascular wall cells expressing APP/PN2. Significant levels of Aβ42 are present in the plasma, suggesting that this reservoir makes a minor contribution to atherosclerotic plaques. Our data reveal that although aortic atherosclerosis and AD cerebrovascular amyloidosis exhibit clearly divergent end-stage manifestations, both vascular diseases share some key pathophysiological promoting elements and pathways. Whether they happen to be deposited in vessels of the central nervous system or atherosclerotic plaques in the periphery, Aβ peptides may promote and perhaps synergize chronic inflammatory processes which culminate in the degeneration, malfunction and ultimate destruction of arterial walls.► Atherosclerotic aortas contained Aβ peptides primarily made of Aβ40 peptide. ► Activated and inactivated platelets revealed that Aβ40 was the dominant peptide. ► Aβ peptides in atherosclerotic lesions mainly originate from activated platelets. ► The likely source of Aβ is vascular wall cells expressing APP/PN2/Aβ and platelets. ► In AVD and AD, Aβ contributes to inflammatory destruction of the vascular wall.
Keywords: Atherosclerosis; Platelet; Amyloid-beta; Vascular inflammation; Alzheimer's disease; Coagulation cascade;

Gene delivery in salivary glands: From the bench to the clinic by Yuval Samuni; Bruce J. Baum (1515-1521).
In vivo gene delivery has long been seen as providing opportunities for the development of novel treatments for disorders refractory to existing therapies. Over the last two decades, salivary glands have proven to be a useful, if somewhat unconventional, target tissue for studying several potential clinical applications of therapeutic gene delivery. Herein, we follow the progress, address some problems and assess the outlook for clinical applications of salivary gland gene delivery. Our experience with these tissues provides a roadmap for the process of moving an idea from the laboratory bench to patients.► This review offers a roadmap of bench to clinic research using salivary gland gene transfer applications as an example. ► Advantages of salivary glands as gene transfer target sites are described. ► A key clinical application (repair of radiation-damaged glands) is emphasized. ► The opportunities and difficulties associated with bench to clinic research are considered.
Keywords: Gene therapy; Salivary gland; Translational research;

The nuclear receptor FXR regulates hepatic transport and metabolism of glutamine and glutamate by Barbara Renga; Andrea Mencarelli; Sabrina Cipriani; Claudio D'Amore; Angela Zampella; Maria Chiara Monti; Eleonora Distrutti; Stefano Fiorucci (1522-1531).
Hepatic transport and metabolism of glutamate and glutamine are regulated by intervention of several proteins. Glutamine is taken up by periportal hepatocytes and is the major source of ammonia for urea synthesis and glutamate for N-acetylglutamate (NAG) synthesis, which is catalyzed by the N-acetylglutamate synthase (NAGS). Glutamate is taken up by perivenous hepatocytes and is the main source for the synthesis of glutamine, catalyzed by glutamine synthase (GS). Accumulation of glutamate and ammonia is a common feature of chronic liver failure, but mechanism that leads to failure of the urea cycle in this setting is unknown. The Farnesoid X Receptor (FXR) is a bile acid sensor in hepatocytes. Here, we have investigated its role in the regulation of the metabolism of both glutamine and glutamate. In vitro studies in primary cultures of hepatocytes from wild type and FXR−/− mice and HepG2 cells, and in vivo studies, in FXR−/− mice as well as in a rodent model of hepatic liver failure induced by carbon tetrachloride (CCl4), demonstrate a role for FXR in regulating this metabolism. Further on, promoter analysis studies demonstrate that both human and mouse NAGS promoters contain a putative FXRE, an ER8 sequence. EMSA, ChIP and luciferase experiments carried out to investigate the functionality of this sequence demonstrate that FXR is essential to induce the expression of NAGS. In conclusion, FXR activation regulates glutamine and glutamate metabolism and FXR ligands might have utility in the treatment of hyperammonemia states.Display Omitted► FXR regulates glutamine and glutamate metabolism genes. ► FXR activation induces urea synthesis and ammonia detoxification. ► FXR activation could be beneficial for the treatment of hyperammonemia in chronic liver diseases.
Keywords: Glutamine; Glutamate; Urea cycle; NAGS; FXR; Hyperammonemia;

Development of diabetes in lean Ncb5or-null mice is associated with manifestations of endoplasmic reticulum and oxidative stress in beta cells by WenFang Wang; Ying Guo; Ming Xu; Han-Hung Huang; Lesya Novikova; Kevin Larade; Zhi-Gang Jiang; Terri C. Thayer; Jennifer R. Frontera; Daniel Aires; Helin Ding; John Turk; Clayton E. Mathews; H. Franklin Bunn; Lisa Stehno-Bittel; Hao Zhu (1532-1541).
NADH-cytochrome b5 oxidoreductase (Ncb5or) is an endoplasmic reticulum (ER)-associated redox enzyme involved in fatty acid metabolism, and phenotypic abnormalities of Ncb5or−/− mice include diabetes and lipoatrophy. These mice are lean and insulin-sensitive but become hyperglycemic at age 7 weeks as a result of β-cell dysfunction and loss. Here we examine early cellular and molecular events associated with manifestations of β-cell defects in Ncb5or−/− mice. We observe lower islet β-cell content in pancreata at age 4 weeks and prominent ER distention in β-cells by age 5 weeks. Ultrastructural changes progress rapidly in severity from age 5 to 6 weeks, and their frequency rises from 10% of β-cells at 5 weeks to 33% at 6 weeks. These changes correlate temporally with the onset of diabetes. ER stress responses and lipid load in Ncb5or−/− β-cells were assessed with isolated islets from mice at age 5 weeks. Expression levels of the stress marker protein Grp78/BiP and of phosphorylated eIF2α protein were found to be reduced, although their transcript levels did not decline. This pattern stands in contrast to the canonical unfolded protein response. Ncb5or−/− β-cells also accumulated higher intracellular levels of palmitate and other free fatty acids and exhibited greater reactive oxygen species production than wild-type cells. An alloxan-susceptible genetic background was found to confer accelerated onset of diabetes in Ncb5or−/− mice. These findings provide the first direct evidence that manifestations of diabetes in lean Ncb5or−/− mice involve saturated free fatty acid overload of β-cells and ER and oxidative stress responses.► We characterize early events in beta-cell dysfunction in a lean diabetic mouse. ► We examine beta-cell lipids, endoplasmic reticulum and oxidative stress. ► Severity and abundance of distended endoplasmic reticulum worsen over time. ► Elevated levels of saturated fatty acids are associated with cellular stress. ► Manifestation of diabetes correlates to endoplasmic reticulum and oxidative stress.
Keywords: Oxidative stress; ER stress; Free fatty acid; Diabetes; Beta-cell;

Bone, joint and tooth development in mucopolysaccharidoses: Relevance to therapeutic options by E. Oussoren; M.M.M.G. Brands; G.J.G. Ruijter; A.T. van der Ploeg; A.J.J. Reuser (1542-1556).
The mucopolysaccharidoses (MPS) are prominent among the lysosomal storage diseases. The intra-lysosomal accumulation of glycosaminoglycans (GAGs) in this group of diseases, which are caused by several different enzyme deficiencies, induces a cascade of responses that affect cellular functions and maintenance of the extra-cellular matrix. Against the background of normal tissue-specific processes, this review summarizes and discusses the histological and biochemical abnormalities reported in the bones, joints, teeth and extracellular matrix of MPS patients and animal models. With an eye to the possibilities and limitations of reversing the pathological changes in the various tissues, we address therapeutic challenges, and present a model in which the cascade of pathologic events is depicted in terms of primary and secondary events.► Bone, joint and tooth anomalies in MPS: derailment of normal developmental processes. ► Therapeutic challenges to reverse pathological changes in MPS tissues. ► A model depicting the primary and secondary pathologic events in MPS.
Keywords: Mucopolysaccharidosis (MPS); Lysosomal storage disorder; Bone; Joint; Tooth;

Genetic modifiers of non-alcoholic fatty liver disease progression by Quentin M. Anstee; Ann K. Daly; Christopher P. Day (1557-1566).
Non-alcoholic fatty liver disease (NAFLD) is now recognised as the most common cause of liver dysfunction worldwide. However, whilst the majority of individuals who exhibit features of the metabolic syndrome including obesity and insulin resistance will develop steatosis, only a minority progress to steatohepatitis, fibrosis and cirrhosis. Subtle inter-patient genetic variations and environment interact to determine disease phenotype and influence progression. A decade after the sequencing of the human genome, the comprehensive study of genomic variation offers new insights into the modifier genes, pathogenic mechanisms and is beginning to suggest novel therapeutic targets. We review the current status of the field with particular focus on advances from recent genome-wide association studies.► NAFLD/NASH is a common, progressive liver disease. ► Subtle inter-patient genetic variation and environment influence disease progression. ► GWAS and candidate gene studies have identified several genetic modifiers. ► Polymorphisms in PNPLA3 have been identified and replicated in several studies. ► Understanding the genetic basis of disease will suggest novel therapeutic targets.
Keywords: NAFLD; NASH; Steatohepatitis; Gene; Polymorphism;