BBA - Molecular Basis of Disease (v.1852, #12)

Lack of cyclophilin D protects against the development of acute lung injury in endotoxemia by Fruzsina Fonai; Janos K. Priber; Peter B. Jakus; Nikoletta Kalman; Csenge Antus; Edit Pollak; Gergely Karsai; Laszlo Tretter; Balazs Sumegi; Balazs Veres (2563-2573).
Sepsis caused by LPS is characterized by an intense systemic inflammatory response affecting the lungs, causing acute lung injury (ALI). Dysfunction of mitochondria and the role of reactive oxygen (ROS) and nitrogen species produced by mitochondria have already been proposed in the pathogenesis of sepsis; however, the exact molecular mechanism is poorly understood. Oxidative stress induces cyclophilin D (CypD)-dependent mitochondrial permeability transition (mPT), leading to organ failure in sepsis. In previous studies mPT was inhibited by cyclosporine A which, beside CypD, inhibits cyclophilin A, B, C and calcineurin, regulating cell death and inflammatory pathways. The immunomodulatory side effects of cyclosporine A make it unfavorable in inflammatory model systems. To avoid these uncertainties in the molecular mechanism, we studied endotoxemia-induced ALI in CypD−/− mice providing unambiguous data for the pathological role of CypD-dependent mPT in ALI. Our key finding is that the loss of this essential protein improves survival rate and it can intensely ameliorate endotoxin-induced lung injury through attenuated proinflammatory cytokine release, down-regulation of redox sensitive cellular pathways such as MAPKs, Akt, and NF-κB and reducing the production of ROS. Functional inhibition of NF-κB was confirmed by decreased expression of NF-κB-mediated proinflammatory genes. We demonstrated that impaired mPT due to the lack of CypD reduces the severity of endotoxemia-induced lung injury suggesting that CypD specific inhibitors might have a great therapeutic potential in sepsis-induced organ failure. Our data highlight a previously unknown regulatory function of mitochondria during inflammatory response.
Keywords: Acute lung injury; Lipopolysaccharide; Cyclophilin D; Reactive oxygen species; NF-κB;

GSK3β and Gli3 play a role in activation of Hedgehog-Gli pathway in human colon cancer — Targeting GSK3β downregulates the signaling pathway and reduces cell proliferation by Diana Trnski; Maja Sabol; Ante Gojević; Marina Martinić; Petar Ozretić; Vesna Musani; Snježana Ramić; Sonja Levanat (2574-2584).
The role of Hedgehog-Gli (Hh-Gli) signaling in colon cancer tumorigenesis has not yet been completely elucidated. Here we provide strong evidence of Hh-Gli signaling involvement in survival of colon cancer cells, with the main trigger of activation being deregulated GSK3β.Our clinical data reveals high expression levels of GSK3β and Gli3 in human colon cancer tissue samples, with positive correlation between GSK3β expression and DUKES' stage. Further experiments on colon cancer cell lines have shown that a deregulated GSK3β upregulates Hh-Gli signaling and positively affects colon cancer cell survival. We show that inhibition of GSK3β with lithium chloride enhances Gli3 processing into its repressor form, consequently downregulating Hh-Gli signaling, reducing cell proliferation and inducing cell death. Analysis of the molecular mechanisms revealed that lithium chloride enhances Gli3–SuFu–GSK3β complex formation leading to more efficient Gli3 cleavage and Hh-Gli signaling downregulation. This work proposes that activation of the Hh-Gli signaling pathway in colon cancer cells occurs non-canonically via deregulated GSK3β. Gli3 seems to be the main pathway effector, highlighting the activator potential of this transcription factor, which is highly dependent on GSK3β function and fine tuning of the Gli3–SuFu–GSK3β platform.
Keywords: Hedgehog signaling; Gli3; GSK3β; Autophagy; Apoptosis; Colon cancer;

The zinc binding receptor GPR39 interacts with 5-HT1A and GalR1 to form dynamic heteroreceptor complexes with signaling diversity by Mercè Tena-Campos; Eva Ramon; Dasiel O. Borroto-Escuela; Kjell Fuxe; Pere Garriga (2585-2592).
GPR39 is a class A G protein-coupled receptor involved in zinc binding and glucose homeostasis regulation, among other physiological processes. GPR39 was originally thought to be the receptor for obestatin peptide but this view has been challenged. However, activation of this receptor by zinc has been clearly established. Recent studies suggest that low GPR39 expression, due to deficient zinc levels, is involved in major depressive disorder. We have previously reported that zinc can alter receptor–receptor interactions and favor specific receptor interactions. In order to unravel the effect of zinc on specific G protein-coupled receptor association processes, we have performed FRET and co-immunopurification studies with GPR39 and 5-HT1A and GalR1 which have been shown to dimerize. Our results suggest that zinc can modulate the formation of specific 5-HT1A-GPR39 and GalR1-5-HT1A-GPR39 heteroreceptor complexes under our experimental conditions.We have analyzed the differences in signaling between the mono-homomeric receptors 5-HT1A, GalR1 and GPR39 and the heteroreceptor complexes between them Our results show that the GPR39-5-HT1A heterocomplex has additive functionalities when compared to the monomeric–homomeric receptors upon receptor activation. In addition, the heterocomplex including also GalR1 shows a different behavior, upon exposure to the same agonists. Furthermore, these processes appear to be regulated by zinc. These findings provide a rationale for the antidepressive effect widely described for zinc because pro-depressive heterocomplexes are predominant at low zinc concentration levels.Display Omitted
Keywords: Depression; G protein-coupled receptor; Heterooligomerization; Purified receptor; Signal transduction; Zinc;

High steady-state reactive oxygen species (ROS) production has been implicated with metastatic disease progression. We provide new evidence that this increased intracellular ROS milieu uniquely predisposes metastatic tumor cells to hypoxia-mediated regulation of the matrix metalloproteinase MMP-1. Using a cell culture metastatic progression model we previously reported that steady-state intracellular H2O2 levels are elevated in highly metastatic 253J-BV bladder cancer cells compared to their non-metastatic 253J parental cells. 253J-BV cells display higher basal MMP-1 expression, which is further enhanced under hypoxic conditions (1% O2). This hypoxia-mediated MMP-1 increase was not observed in the non-metastatic 253J cells. Hypoxia-induced MMP-1 increases are accompanied by the stabilization of hypoxia-inducible transcription factors (HIFs)-1α and HIF-2α, and a rise in intracellular ROS in metastatic 253J-BV cells. RNA interference studies show that hypoxia-mediated MMP-1 expression is primarily dependent on the presence of HIF-2α. Further, hypoxia promotes migration and spheroid outgrowth of only the metastatic 253J-BV cells and not the parental 253J cells. The observed HIF stabilization, MMP-1 expression and migration under hypoxia are dependent on increases in intracellular ROS, as these effects are attenuated by treatment with the antioxidant N-acetyl-L-cysteine. These data show that ROS play an important role in hypoxia-mediated MMP-1 expression and that an elevated intracellular redox environment, as observed in metastasis, predisposes tumor cells to an enhanced hypoxic response. It further supports the notion that metastatic tumor cells are uniquely able to utilize intracellular increases in ROS to drive pro-metastatic signaling events and highlights the important interplay between ROS and hypoxia in malignancy.
Keywords: Hypoxia; MMP-1; Reactive oxygen species; H2O2; HIF-2α; Metastatic bladder cancer;

Molecular changes associated with chronic liver damage and neoplastic lesions in a murine model of hereditary tyrosinemia type 1 by Francesca Angileri; Vincent Roy; Geneviève Morrow; Jean Yves Scoazec; Nicolas Gadot; Diana Orejuela; Robert M. Tanguay (2603-2617).
Hereditary tyrosinemia type 1 (HT1) is the most severe inherited metabolic disease of the tyrosine catabolic pathway, with a progressive hepatic and renal injury and a fatal outcome if untreated. Toxic metabolites accumulating in HT1 have been shown to elicit endoplasmic reticulum (ER) stress response, and to induce chromosomal instability, cell cycle arrest and apoptosis perturbation. Although many studies have concentrated on elucidating these events, the molecular pathways responsible for development of hepatocellular carcinoma (HCC) still remain unclear. In this study the fah knockout murine model (fah / ) was used to investigate the cellular signaling implicated in the pathogenesis of HT1. Fah / mice were subjected to drug therapy discontinuation (Nitisinone withdrawal), and livers were analyzed at different stages of the disease. Monitoring of mice revealed an increasing degeneration of the overall physiological conditions following drug withdrawal. Histological analysis unveiled diffuse hepatocellular damage, steatosis, oval-like cells proliferation and development of liver cell adenomas. Immunoblotting results revealed a progressive and chronic activation of stress pathways related to cell survival and proliferation, including several stress regulators such as Nrf2, eIF2α, CHOP, HO-1, and some members of the MAPK signaling cascade. Impairment of stress defensive mechanisms was also shown by microarray analysis in fah / mice following prolonged therapy interruption. These results suggest that a sustained activation of stress pathways in the chronic HT1 progression might play a central role in exacerbating liver degeneration.
Keywords: ER stress; Hereditary tyrosinemia type 1 (HT1); Metabolic diseases; Hepatoma; Liver dysfunction; HO-1;

Retinal pericyte loss and neovascularization are characteristic features of diabetic retinopathy. Gemigliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic retinopathy have not yet been reported. We evaluated the efficacy of gemigliptin on retinal vascular leakage in db/db mice, which is an animal model for type 2 diabetes, and neovascularization in oxygen-induced retinopathy (OIR) mice, which is an animal model for ischemic proliferative retinopathy. Gemigliptin (100 mg/kg/day) was orally administered to the db/db mice for 12 weeks. C57BL/6 mice on postnatal day 7 (P7) were exposed to 75% hyperoxia for 5 days, followed by exposure to room air from P12 to P17 to induce OIR. Gemigliptin (50 mg/kg/day) was intraperitoneally injected daily from P12 to P17. Retinal neovascularization was analyzed in flat-mounted retinas on P17. We determined the efficacy and possible mechanism of gemigliptin on high glucose-induced apoptosis of primary human retinal pericytes. The oral administration of gemigliptin for 4 months significantly ameliorated retinal pericyte apoptosis and vascular leakage in the db/db mice. Gemigliptin also ameliorated retinal neovascularization in the OIR mice. Gemigliptin attenuated the overexpression of plasminogen activator inhibitor-1 (PAI-1) in the retinas of diabetic and OIR mice. Gemigliptin and PAI-1 siRNA significantly inhibited pericyte apoptosis by inhibiting the overexpression of PAI-1, which is induced by high glucose. Our results suggest that gemigliptin has potent anti-angiogenic and anti-apoptotic activities via suppressing DPP-4 and PAI-1, and the results support the direct retinoprotective action of gemigliptin.
Keywords: Diabetic retinopathy; Gemigliptin; Neovascularization; Pericyte;

The increased potassium intake improves cognitive performance and attenuates histopathological markers in a model of Alzheimer's disease by Pedro Cisternas; Carolina B. Lindsay; Paulina Salazar; Carmen Silva-Alvarez; Rocio M. Retamales; Felipe G. Serrano; Carlos P. Vio; Nibaldo C. Inestrosa (2630-2644).
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by hallmarks that include an accumulation of amyloid-β peptide (Aβ), inflammation, oxidative stress and synaptic dysfunction, which lead to a decrease in cognitive function. To date, the onset and progression of AD have been associated with pathologies such as hypertension and diabetes. Hypertension, a disease with a high incidence worldwide, is characterized by a chronic increase in blood pressure. Interestingly, this disease has a close relationship to the eating behavior of patients because high Na+ intake is a significant risk factor for hypertension. In fact, a decrease in Na+ consumption, along with an increase in K+ intake, is a primary non-pharmacological approach to preventing hypertension. In the present work, we examined whether an increase in K+ intake affects the expression of certain neuropathological markers or the cognitive performance of a murine model of AD. We observed that an increase in K+ intake leads to a change in the aggregation pattern of the Aβ peptide, a partial decrease in some epitopes of tau phosphorylation and improvement in the cognitive performance. The recovery in cognitive performance was correlated with a significant improvement in the generation of long-term potentiation. We also observed a decrease in markers related to inflammation and oxidative stress such as glial fibrillary acidic protein (GFAP), interleukin 6 (IL-6) and 4-hydroxynonenal (4-HNE). Together, our data support the idea that changes in diet, such as an increase in K+ intake, may be important in the prevention of AD onset as a non-pharmacological therapy.
Keywords: Alzheimer's disease; Potassium intake; Hypertension; Synaptic dysfunction;

Expansion size and presence of CCG/CTC/CGG sequence interruptions in the expanded CTG array are independently associated to hypermethylation at the DMPK locus in myotonic dystrophy type 1 (DM1) by Massimo Santoro; Luana Fontana; Marcella Masciullo; Maria Laura Ester Bianchi; Salvatore Rossi; Emanuele Leoncini; Giuseppe Novelli; Annalisa Botta; Gabriella Silvestri (2645-2652).
A differential CpG methylation profile upstream of the expanded CTG array at the DMPK locus has been reported in patients with myotonic dystrophy type 1 (DM1), suggesting that hypermethylation might modulate DM1 phenotype, possibly affecting expression levels of DMPK and/or flanking genes. To clarify this issue, we characterized by methylation sensitive high resolution melting (MS-HRM) the CpG methylation pattern of DNA sequences flanking the pathological CTG expansion in 13 childhood-onset, 37 juvenile/adult-onset, 7 congenital DM1 patients carrying uninterrupted CTG expansions and in 9 DM1 patients carrying variant expansions vs 30 controls. Association of methylation status with disease features (nCTG, age, sex, MIRS, disease duration) was also assessed. Finally, DMPK and SIX5 expression levels were evaluated in leukocytes from controls, methylated and unmethylated DM1 patients.We found hypermethylation involving upstream sequences of DM1 locus in patients with uninterrupted CTG expansions > 1000 CTG and affected by a congenital or childhood onset form. Besides the n(CTG) and early disease onset, hypermethylation was also significantly associated with maternal transmission.On the other hand, hypermethylation involved the 3′ of the CTG array in DM1 patients carrying variant expansions. DMPK and SIX5 expression did not significantly differ in methylated vs unmethylated DM1 patients. Our results suggest that either the inherited size of the expanded allele and the presence of interruptions at the 3′ end are associated with a highly polarized pattern of CpG methylation at the DM1 locus and that, at least in leukocytes, DM1 locus hypermethylation would not significantly affect DMPK or SIX5 expression.
Keywords: Myotonic dystrophy type 1; Variant expansions; Methylation; High resolution melting; DMPK; SIX5;

Targeting the interface of the pathological complex of α-synuclein and TPPP/p25 by Sándor Szunyogh; Judit Oláh; Tibor Szénási; Adél Szabó; Judit Ovádi (2653-2661).
The pathological interaction of intrinsically disordered proteins, such as α-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25), is often associated with neurodegenerative disorders. These hallmark proteins are co-enriched and co-localized in brain inclusions of Parkinson's disease and other synucleinopathies; yet, their successful targeting does not provide adequate effect due to their multiple functions. Here we characterized the interactions of the human recombinant wild type SYN, its truncated forms (SYN1–120, SYN95–140), a synthetized peptide (SYN126–140) and a proteolytic fragment (SYN103–140) with TPPP/p25 to identify the SYN segment involved in the interaction. The binding of SYN103–140 to TPPP/p25 detected by ELISA suggested the involvement of a segment within the C-terminal of SYN. The studies performed with ELISA, Microscale Thermophoresis and affinity chromatography proved that SYN95–140 and SYN126–140 – in contrast to SYN1–120 – displayed significant binding to TPPP/p25. Fluorescence assay with ANS, a molten globule indicator, showed that SYN, but not SYN1–120 abolished the zinc-induced local folding of both the full length as well as the N- and C-terminal-free (core) TPPP/p25; SYN95–140 and SYN126–140 were effective as well. The aggregation-prone properties of the SYN species with full length or core TPPP/p25 visualized by immunofluorescent microscopy demonstrated that SYN95–140 and SYN126–140, but not SYN1–120, induced co-enrichment and massive intracellular aggregation after their premixing and uptake from the medium. These data with their innovative impact could contribute to the development of anti-Parkinson drugs with unique specificity by targeting the interface of the pathological TPPP/p25-SYN complex.
Keywords: α-Synuclein; Binding motives; Neomorphic moonlighting function; TPPP/p25; Parkinson's disease;

Inhibition of cereblon by fenofibrate ameliorates alcoholic liver disease by enhancing AMPK by Yong Deuk Kim; Kwang Min Lee; Seung-Lark Hwang; Hyeun Wook Chang; Keuk-Jun Kim; Robert A. Harris; Hueng-Sik Choi; Won-Sik Choi; Sung-Eun Lee; Chul-Seung Park (2662-2670).
Alcohol consumption exacerbates alcoholic liver disease by attenuating the activity of AMP-activated protein kinase (AMPK). AMPK is activated by fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, and inhibited by direct interaction with cereblon (CRBN), a component of an E3 ubiquitin ligase complex. Based on these preliminary findings, we investigated that CRBN would be up-regulated in the liver by alcohol consumption and that CRBN deficiency would ameliorate hepatic steatosis and pro-inflammatory responses in alcohol-fed mice by increasing AMPK activity. Wild-type, CRBN and PPARα null mice were fed an alcohol-containing liquid diet and administered with fenofibrate. Gene expression profiles and metabolic changes were measured in the liver and blood of these mice. Expression of CRBN, cytochrome P450 2E1 (CYP2E1), lipogenic genes, pro-inflammatory cytokines, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were increased in the Lieber–DeCarli alcohol-challenged mice. Fenofibrate attenuated the induction of CRBN and reduced hepatic steatosis and pro-inflammatory markers in these mice. Ablation of the gene encoding CRBN produced the same effect as fenofibrate. The increase in CRBN gene expression by alcohol and the reduction of CRBN expression by fenofibrate were negated in PPARα null mice. Fenofibrate increased the recruitment of PPARα on CRBN gene promoter in WT mice but not in PPARα null mice. Silencing of AMPK prevented the beneficial effects of fenofibrate. These results demonstrate that activation of PPARα by fenofibrate alleviates alcohol-induced hepatic steatosis and inflammation by reducing the inhibition of AMPK by CRBN. CRBN is a potential therapeutic target for the alcoholic liver disease.
Keywords: Cytokine; Gene expression; Inflammation; PPARα; Steatosis;

Fatty acids (FAs) are massively released from peripancreatic fat during acute pancreatitis (AP) and they were shown, as a whole, to induce inflammatory response in pancreatic acini. We investigated the mechanisms triggered by the major saturated FAs (SFAs) and unsaturated FAs (UFAs) in modulating the expression of chemokine (C–C motif) ligand 2 (CCL2) in acinar cells. Pancreatic acini of control rats were treated with palmitic acid (PA) or stearic acid (SA), as SFAs, or oleic acid (OA) or linoleic acid (LA), as UFAs. By using specific inhibitors, the involvement of MAPKs (JNK, ERK, p38), JAK, NF-κB and STAT3 pathways was assessed. The role of PPARγ pathway was studied by using 15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ2). CCL2 mRNA was analyzed by qRT-PCR. By western blot, phosphorylated forms of MAPKs and JAK as well as IκB-α were analyzed in cytoplasm and p65-NF-κB and phospho-STAT3 in nucleus. No effect was found in PA- or SA-treated acini. Conversely, in response to OA or LA, MAPKs and JAK acted as upstream signals, driving the CCL2 up-regulation transcriptionally mediated by the synergic action of NF-κB and STAT3. By blocking the activation of NF-κB and STAT3, 15d-PGJ2 totally inhibited the OA- and LA-induced CCL2 overexpression. We conclude that the most common UFAs, but not the SFAs, represented in peripancreatic fat and released during AP, are capable of up-regulating the acinar expression of CCL2, which depends on the activation of MAPK/JAK-mediated NF-κB and STAT3 pathways. By targeting both transcription factors, PPARγ agonists could be indicated as potential therapy in AP.
Keywords: CCL2; Inflammation; Pancreatic acini; Saturated fatty acids; Unsaturated fatty acids;

Elevated protein arginine methyltransferase 1 expression regulates fibroblast motility in pulmonary fibrosis by Dariusz Zakrzewicz; Anna Zakrzewicz; Miroslava Didiasova; Marek Korencak; Djuro Kosanovic; Ralph T. Schermuly; Philipp Markart; Malgorzata Wygrecka (2678-2688).
Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by epithelial cell injury, fibroblast activation and excessive extracellular matrix deposition. Although protein arginine methyltransferase 1 (PRMT1) was found to regulate cell proliferation, differentiation and migration, its role in the development/progression of IPF has not yet been described.Expression of PRMT1 was elevated in lung homogenates from IPF patients. Significant upregulation of PRMT1 expression was also observed in the lungs of bleomycin-treated mice. Immunohistochemical analysis revealed PRMT1-positive staining in fibroblasts/myofibroblasts and alveolar type II cells of IPF lungs and in fibrotic lesions of bleomycin-injured lungs. Fibroblasts isolated from IPF lungs demonstrated increased PRMT1 expression. Interleukin-4 (IL-4), a profibrotic cytokine, enhanced the expression of PRMT1 and the migration of donor and IPF fibroblasts. Interference with the expression or the activity of PRMT1 diminished the migration of the cells in response to IL-4. Strikingly, even though the incubation of donor and IPF fibroblasts with IL-4 did not affect their proliferation, depletion, but not blockage of PRMT1 activity suppressed cell growth.PRMT1 can contribute to the development of pulmonary fibrosis by regulating fibroblast activities. Thus, interference with its expression and/or activity may provide a novel therapeutic option for patients with IPF.
Keywords: ADMA; Arginine methylation; Fibroblasts; Interleukin-4; Migration; PRMT; Proliferation;

Lack of phosphatidylethanolamine N-methyltransferase alters hepatic phospholipid composition and induces endoplasmic reticulum stress by Xia Gao; Jelske N. van der Veen; Jean E. Vance; Aducio Thiesen; Dennis E. Vance; René L. Jacobs (2689-2699).
Endoplasmic reticulum (ER) stress is associated with development of steatohepatitis. Phosphatidylethanolamine N-methyltransferase (PEMT) is a hepatic enzyme located on the ER and mitochondria-associated membranes and catalyzes phosphatidylcholine (PC) synthesis via methylation of phosphatidylethanolamine (PE). We hypothesized that PEMT deficiency in mice alters ER phospholipid content, thereby inducing ER stress and sensitizing the mice to diet-induced steatohepatitis.PC and PE mass were measured in hepatic ER fractions from chow-fed and high fat-fed Pemt−/− and Pemt+/+ mice. Proteins implicated in ER stress and the unfolded protein response (UPR) were assessed in mouse livers and in McArdle-RH7777 hepatoma cells that expressed or lacked PEMT. The chemical chaperone 4-phenyl butyric acid was administered to cells and HF-fed Pemt−/− mice to alleviate ER stress.In chow-fed Pemt−/− mice, the hepatic PC/PE ratio in the ER was lower than in Pemt+/+ mice, and levels of ER stress markers, CHOP and BIP, were higher without activation of the UPR. In livers of HF-fed Pemt−/− mice the ER had a lower PC/PE ratio, and exhibited more ER stress and UPR activation. Similarly, the UPR was repressed in McArdle cells expressing PEMT compared with those lacking PEMT, with concomitantly lower levels of CHOP and BIP. 4-Phenyl butyric acid attenuated activation of the UPR and ER stress in McArdle cells lacking PEMT, but not the hepatic ER stress in HF-fed Pemt−/− mice.PEMT deficiency reduces the PC/PE ratio in the ER and induces ER stress, which sensitizes the mice to HF-induced steatohepatitis.
Keywords: Steatohepatitis; Unfolded protein response; Phenylbutyric acid; Phosphatidylcholine; Phosphatidylethanolamine; ER stress;

Hydroxyproline metabolism in a mouse model of Primary Hyperoxaluria Type 3 by Xingsheng Li; John Knight; W. Todd Lowther; Ross P. Holmes (2700-2705).
Primary Hyperoxaluria Type 3 is a recently discovered form of this autosomal recessive disease that results from mutations in the gene coding for 4-hydroxy-2-oxoglutarate aldolase (HOGA1). This enzyme is one of the 2 unique enzymes in the hydroxyproline catabolism pathway. Affected individuals have increased urinary excretions of oxalate, 4-hydroxy-L-glutamate (4-OH-Glu), 4-hydroxy-2-oxoglutarate (HOG), and 2,4-dihydroxyglutarate (DHG). While 4-OH-Glu and HOG are precursor substrates of HOGA1 and increases in their concentrations are expected, how DHG is formed and how HOG to oxalate are unclear. To resolve these important questions and to provide insight into possible therapeutic avenues for treating this disease, an animal model of the disease would be invaluable. We have developed a mouse model of this disease which has null mutations in the Hoga1 gene and have characterized its phenotype. It shares many characteristics of the human disease, particularly when challenged by the inclusion of hydroxyproline in the diet. An increased oxalate excretion is not observed in the KO mice which may be consistent with the recent recognition that only a small fraction of the individuals with the genotype for HOGA deficiency develop PH.
Keywords: Primary Hyperoxaluria; Oxalate; Hydroxyproline; Mouse model;

The role of S100 proteins and their receptor RAGE in pancreatic cancer by Estelle Leclerc; Stefan W. Vetter (2706-2711).
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with low survival rates. Current therapeutic treatments have very poor response rates due to the high inherent chemoresistance of the pancreatic-cancer cells. Recent studies have suggested that the receptor for advanced glycation end products (RAGE) and its S100 protein ligands play important roles in the progression of PDAC. We will discuss the potential role of S100 proteins and their receptor, RAGE, in the development and progression of pancreatic cancer.
Keywords: RAGE; S100 proteins; Pancreatic ductal adenocarcinoma;

Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II by Liliana Matos; Vânia Gonçalves; Eugénia Pinto; Francisco Laranjeira; Maria João Prata; Peter Jordan; Lourdes R. Desviat; Belén Pérez; Sandra Alves (2712-2721).
Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C > T and c.257C > T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C > T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3′ splice site of exon 3 respectively. For the c.1122C > T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n = 3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5′ splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation.
Keywords: Lysosomal storage disorders; IDS gene; Splicing regulation; Antisense therapy;

Signaling pathways underlying skeletal muscle wasting in experimental pulmonary arterial hypertension by Daniel Moreira-Gonçalves; Ana Isabel Padrão; Rita Ferreira; Joana Justino; Rita Nogueira-Ferreira; Maria João Neuparth; Rui Vitorino; Hélder Fonseca; Ana Filipa Silva; José Alberto Duarte; Adelino Leite-Moreira; Tiago Henriques-Coelho (2722-2731).
Skeletal muscle wasting contributes to the poor functional status and quality of life of patients with pulmonary arterial hypertension (PAH). The present study aims to characterize the molecular mechanism underlying skeletal muscle wasting in experimental PAH induced by monocrotaline (MCT).Male Wistar rats were randomly injected with saline solution (CONT; n = 10) or MCT (MCT; 60 mg/kg, s.c.; n = 15). After 4 weeks of MCT or vehicle administration, animals were anesthetized and submitted to right ventricular (RV) hemodynamic evaluation. Blood and gastrocnemius samples were collected and stored for analysis.MCT group developed PAH (70% increase in RV peak systolic pressure) RV dysfunction (increased end-diastolic pressure and Tau), and body and muscle wasting (reduction of 20%, 16% and 30% on body weight, gastrocnemius mass and fiber cross sectional area, respectively). Muscle atrophy was associated with a decrease in type I MHC. Circulating (C reactive protein, myostatin and IL-1beta) and local catabolic markers (MAFbx/atrogin-1, protease activity) were increased in MCT animals, while Akt/mTOR pathway was preserved. Mitochondria isolated from gastrocnemius of MCT animals showed decreased activity of ATP synthase, lower levels of Tfam, accumulation of oxidatively modified proteins together with reduced levels of paraplegin.Our data suggests an anabolic/catabolic imbalance in gastrocnemius from MCT-induced PAH rats. Accumulation of dysfunctional mitochondria due to the inefficiency of protein quality control systems to eliminate damaged proteins could also contribute to muscle atrophy in PAH.
Keywords: Pulmonary arterial hypertension; Skeletal muscle wasting; Proteolysis; Mitochondrial dysfunction;