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BBA - Molecular Basis of Disease (v.1822, #6)

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Brain IL-6 elevation causes neuronal circuitry imbalances and mediates autism-like behaviors by Hongen Wei; Kathryn K. Chadman; Daniel P. McCloskey; Ashfaq M. Sheikh; Mazhar Malik; W. Ted Brown; Xiaohong Li (pp. 831-842).

Abnormal immune responses have been reported to be associated with autism. A number of studies showed that cytokines were increased in the blood, brain, and cerebrospinal fluid of autistic subjects. Elevated IL-6 in autistic brain has been a consistent finding. However, the mechanisms by which IL-6 may be involved in the pathogenesis of autism are not well understood. Here we show that mice with elevated IL-6 in the brain display many autistic features, including impaired cognitive abilities, deficits in learning, abnormal anxiety traits and habituations, as well as decreased social interactions. IL-6 elevation caused alterations in excitatory and inhibitory synaptic formations and disrupted the balance of excitatory/inhibitory synaptic transmissions. IL-6 elevation also resulted in an abnormal change in the shape, length and distributing pattern of dendritic spines. These findings suggest that IL-6 elevation in the brain could mediate autistic-like behaviors, possibly through the imbalances of neural circuitry and impairments of synaptic plasticity.►Mice with elevated IL-6 in the brain display many autistic features. ►IL-6 elevation caused alterations in excitatory and inhibitory synaptic formations. ►IL-6 elevation disrupted the balance of excitatory/inhibitory synaptic transmissions. ►IL-6 elevation impaired the development and maturation of dendritic spines.

Keywords: Autism; Cytokine; IL-6; Synapse development; Synaptic transmission; Autistic-like behavior

TNF-α-induced down-regulation of CDX2 suppresses MEP1A expression in colitis by Mehmet Coskun; Anders Krüger Olsen; Thomas Lindebo Holm; Peter Helding Kvist; Ole Haagen Nielsen; Lene Buhl Riis; Jørgen Olsen; Jesper Thorvald Troelsen (pp. 843-851).

Background/aims: High levels of pro-inflammatory cytokines are linked to inflammatory bowel disease (IBD). The transcription factor Caudal-related homeobox transcription factor 2 (CDX2) plays a crucial role in differentiation of intestinal epithelium and regulates IBD-susceptibility genes, including meprin 1A (MEP1A). The aim was to investigate the expression of CDX2 and MEP1A in colitis; to assess if they are regulated by tumor necrosis factor-α (TNF-α), and finally to reveal if CDX2 is involved in a TNF-α-induced down-regulation of MEP1A. Methods: Expression of CDX2 and MEP1A was investigated in colonic biopsies of ulcerative colitis (UC) patients and in dextran sodium sulfate (DSS)-induced colitis. CDX2 protein expression was investigated by immunoblotting and immunohistochemical procedures. CDX2 and MEP1A regulation was examined in TNF-α-treated Caco-2 cells by reverse transcription-polymerase chain reaction and with reporter gene assays, and the effect of anti-TNF-α treatment was assessed using infliximab. Finally, in vivo CDX2–DNA interactions were investigated by chromatin immunoprecipitation. Results: The CDX2 and MEP1A mRNA expression was significantly decreased in active UC patients and in DSS-colitis. Colonic biopsy specimens from active UC showed markedly decreased CDX2 staining. TNF-α treatment diminished the CDX2 and MEP1A mRNA levels, a decrease which, was counteracted by infliximab treatment. Reporter gene assays showed significantly reduced CDX2 and MEP1A activity upon TNF-α stimulation. Finally, TNF-α impaired the ability of CDX2 to interact and activate its own, as well as the MEP1A expression. Conclusions: The present results indicate that a TNF-α-mediated down-regulation of CDX2 can be related to suppressed expression of MEP1A during intestinal inflammation.► CDX2 regulates MEP1A expression in intestinal epithelial cells. ► CDX2 and MEP1A expression are correlatively decreased in intestinal inflammation. ► High levels of TNF-α suppresses the ability of CDX2 to activate MEP1A expression. ► A dysfunction of CDX2 is a key contributor in the pathogenesis of IBD.

Keywords: Abbreviations; CD; Crohn's disease; CDX2; Caudal; -related homeobox transcription factor 2; ChIP; chromatin immunoprecipitation; CK20; cytokeratin 20; CLDN2; claudin-2; DAI; disease activity index; DSS; dextran sodium sulfate; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GSK3β; glycogen synthase kinase-3β; HA; hemagglutinin; HNF4α; hepatocyte nuclear factor 4 alpha; IBD; inflammatory bowel disease; IEC; intestinal epithelial cell; IFX; infliximab; IHC; immunohistochemistry; INF-γ; interferon-γ; MEP1A; meprin 1A; MUC2; mucin 2; NF; nuclear factor; PEPT1; peptide transporter 1; qRT-PCR; quantitative reverse-transcription polymerase chain reaction; RPLP0; ribosomal protein large p0; TNF-α; tumor necrosis factor-α; UC; ulcerative colitisDSS-colitis; Inflammatory bowel disease; Infliximab; Intestine; Tumor necrosis factor; Ulcerative colitis

Mitochondrial reactive oxygen species mediates nicotine-induced hypoxia-inducible factor-1α expression in human non-small cell lung cancer cells by Lili Guo; Lin Li; Weiqiang Wang; Zhenhua Pan; Qinghua Zhou; Zhihao Wu (pp. 852-861).

Cigarette smoking is not only a documented risk for lung carcinogenesis but also promotes lung cancer development. Nicotine, a major component of cigarette smoke but not a carcinogen by itself, has been found to induce proliferation, invasion and metastasis of non-small cell lung cancer (NSCLC). Here we reported that proinvasive effect of nicotine is analogous to that of hypoxia and involves stabilization and activation of hypoxia-inducible factor (HIF)-1α, a key factor in determining the presence of HIF-1 and expression of its downstream metastasis-associated genes. Furthermore, nicotine-induced upregulation of HIF-1α was dependent on mitochondria-derived reactive oxygen species (ROS). Ecotopic expression of mitochondrial targeted catalase effectively prevented nicotine-induced accumulation of HIF-1α protein. In addition, we demonstrated that the effect of nicotine in upregulation of HIF-1α was mediated by Dihydro-β-erythroidine (DhβE)-sensitive nicotine acetylcholine receptors (nAChRs) and required synergistic cooperation of Akt and mitogen-activated protein kinase (MAPK) pathways. These results suggest that exposure to nicotine could mimic effects of hypoxia to stimulate HIF-1α accumulation and activity that might underlie the high metastatic potential of lung cancer.► We reported that nicotine-induced HIF-1α was dependent on mitochondria-derived ROS. ► The effect of nicotine was mediated by DhβE-sensitive nicotine receptors. ► Nicotine-induced HIF-1α required cooperation of Akt and MAPK pathways.

Keywords: Nicotine; Hypoxia-inducible factor 1α; Mitochondria; Reactive oxygen species; Lung cancer metastasis

Dynamin-related protein 1 heterozygote knockout mice do not have synaptic and mitochondrial deficiencies by Maria Manczak; Hiromi Sesaki; Yusuke Kageyama; P. Hemachandra Reddy (pp. 862-874).

The objective of this study was to elucidate the effect of partial reduction of the mitochondrial fission protein, dynamin-related protein 1 (Drp1) on mitochondrial activity and synaptic viability. Recent knockout studies of Drp1 revealed that homozygote Drp1 knockout mice are embryonic lethal due to reduced mitochondrial fission, and that this reduced fission leads to developmental defects in the brain. In contrast, heterozygote Drp1 knockout mice appear to be normal in terms of lifespan, fertility, and viability, and phenotypically these animals are not different from wild-type mice. However, the effects of partial Drp1 reduction on mitochondrial function and synaptic activity are not well understood. In the present study, we sought to characterize synaptic, dendritic and mitochondrial proteins, and mitochondrial function and GTPase enzymatic activity, in Drp1 heterozygote knockout mice. Interestingly, we found no significant changes in synaptic, dendritic, and mitochondrial proteins in the Drp1 heterozygote knockout mice compared to the wild-type mice. Further, mitochondrial function and GTPase enzymatic activity appeared to be normal. However, H₂O₂ and lipid peroxidation levels were significantly reduced in the Drp1 heterozygote knockout mice compared to the wild-type mice. These findings suggest that partial Drp1 reduction does not affect mitochondrial and synaptic viability and may have therapeutic use in treating patients with Alzheimer's disease and Huntington's disease.► We investigated mitochondrial activity and synaptic viability in Drp1+/− and Drp1+/+ mice. ► No significant changes were found in synaptic, dendritic, and mitochondrial proteins in Drp1+/− mice compared to Drp1+/+ mice. ► Mitochondrial function and GTPase Drp1 enzymatic activity appeared to be normal. ► Free radicals and lipid peroxidation levels were significantly reduced in the Drp1+/− mice compared to Drp1+/+ mice.

Keywords: Dynamin-related protein 1; Mitochondria; Oxidative stress; Alzheimer's disease; Huntington's disease; Mitochondrial dynamics

IQGAP2, A candidate tumour suppressor of prostate tumorigenesis by Yanyun Xie; Judy Yan; Jean-Claude Cutz; Adrian P. Rybak; Lizhi He; Fengxiang Wei; Anil Kapoor; Valentina A. Schmidt; Lijian Tao; Damu Tang (pp. 875-884).

Loss of IQGAP2 contributes to the tumorigenesis of hepatocellular carcinoma and gastric cancer. However, whether IQGAP2 also suppresses prostate tumorigenesis remains unclear. We report here that IQGAP2 is a candidate tumour suppressor of prostate cancer (PC). Elevated IQGAP2 was detected in prostatic intraepithelial neoplasia (PIN), early stages of PCs (Gleason score ≤3), and androgen-dependent LNCaP PC cells. However, IQGAP2 was expressed at substantially reduced levels not only in prostate glands and non-tumorigenic BPH-1 prostate epithelial cells but also in advanced (Gleason score 4 or 5) and androgen-independent PCs. Furthermore, xenograft tumours that were derived from stem-like DU145 cells displayed advanced features and lower levels of IQGAP2 in comparison to xenograft tumours that were produced from non stem-like DU145 cells. Collectively, these results suggest that IQGAP2 functions in the surveillance of prostate tumorigenesis. Consistent with this concept, ectopic IQGAP2 reduced the proliferation of DU145, PC3, and 293T cells as well as the invasion ability of DU145 cells. While ectopic IQGAP2 up-regulated E-cadherin in DU145 and PC3 cells, knockdown of IQGAP2 reduced E-cadherin expression. In primary PC and DU145 cells-derived xenograft tumours, the majority of tumours with high levels of IQGAP2 were strongly-positive for E-cadherin. Therefore, IQGAP2 may suppress PC tumorigenesis, at least in part, by up-regulation of E-cadherin. Mechanistically, overexpression of IQGAP2 significantly reduced AKT activation in DU145 cells and inhibition of AKT activation upregulated E-cadherin, suggesting that IQGAP2 increases E-cadherin expression by inhibiting AKT activation. Taken together, we demonstrate here that IQGAP2 is a candidate tumour suppressor of PC.►We demonstrate IQGAP2 being a candidate of new tumour suppressor of prostate cancer. ►IQGAP2 inhibits the growth and invasion of prostate cancer cells in vitro. ►IQGAP2 increases E-cadherin expression in prostate cancer cells via inhibiting AKT. ►We observed changes in IQGAP2 expression in primary prostate cancer. ►Reduction of IQGAP2 associate with prostate cancer progression.

Keywords: IQGAP2; Tumour suppressor; EMT; AKT; Prostate cancer

Oxidative stress increases BACE1 protein levels through activation of the PKR-eIF2α pathway by François Mouton-Liger; Claire Paquet; Julien Dumurgier; Constantin Bouras; Laurent Pradier; Françoise Gray; Jacques Hugon (pp. 885-896).

Beta-site APP cleaving enzyme 1 (BACE1) is the rate limiting enzyme for accumulation of amyloid β (Aβ)-peptide in the brain in Alzheimer's disease (AD). Oxidative stress (OS) that leads to metabolic dysfunction and apoptosis of neurons in AD enhances BACE1 expression and activity. The activation of c-jun N-terminal kinase (JNK) pathway was proposed to explain the BACE1 mRNA increase under OS. However, little is known about the translational control of BACE1 in OS. Recently, a post-transcriptional increase of BACE1 level controlled by phosphorylation of eIF2α (eukaryotic translation initiation factor-2α) have been described after energy deprivation. PKR (double-stranded RNA dependant protein kinase) is a pro-apoptotic kinase that phosphorylates eIF2α and modulates JNK activation in various cellular stresses. We investigated the relations between PKR, eIF2α and BACE1 in AD brains in APP/PS1 knock-in mice and in hydrogen peroxide-induced OS in human neuroblastoma (SH-SY5Y) cell cultures. Immunoblotting results showed that activated PKR (pPKR) and activated eIF2α (peIF2α) and BACE1 levels are increased in AD cortices and BACE1 correlate with phosphorylated eIF2α levels. BACE1 protein levels are increased in response to OS in SH-SY5Y cells and specific inhibitions of PKR-eIF2α attenuate BACE1 protein levels in this model. Our findings provide a new translational regulation of BACE1, under the control of PKR in OS, where eIF2α phosphorylation regulates BACE1 protein expression.► Activated PKR and eIF2α and BACE1 levels are increased in Alzheimer disease brains. ► BACE1 correlate with eIF2α levels in Alzheimer disease cortices. ► BACE1 protein levels are increased in response to oxidative stress in SH-SY5Y cells. ► Specific inhibitions of PKR-eIF2α attenuate BACE1 protein levels. ► Our findings provide a new translational regulation of BACE1 expression.

Keywords: Alzheimer's disease; BACE1; PKR; eIF2α; Oxidative stress; Neuroblastoma cell cultures

MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction by Janine M. Wilkinson; Rose K. Davidson; Tracey E. Swingler; Eleanor R. Jones; Anthony N. Corps; Phillip Johnston; Graham P. Riley; Adrian J. Chojnowski; Ian M. Clark (pp. 897-905).

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and ‘a disintegrin and metalloprotease domain with thrombospondin motifs’, ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.► MMP and TIMP expression was measured in Dupuytren's disease FPCL assays. ► Collagen-degrading MMPs were individually knocked down in the FPCL using siRNA. ► Knockdown of MMP-2 and MMP-14 inhibited cell-mediated contraction. ► Knockdown of MMP-1 potentiated cell-mediated contraction. ► Knockdown of individual MMPs had no effect on collagen degradation.

Keywords: Abbreviations; ADAMTS; a disintegrin and metalloproteinase domain with thrombospondin motifs; DD; Dupuytren's disease; FPCL; fibroblast-populated collagen lattice; MMP; matrix metalloproteinaseDupuytren's disease; MMP; Collagen; Contraction; FPCL

Toxic effects of expanded ataxin-1 involve mechanical instability of the nuclear membrane by Lisa Mapelli; Claudio Canale; Daniela Pesci; Stefania Averaimo; Fabiana Guizzardi; Valentina Fortunati; Laura Falasca; Mauro Piacentini; Alessandra Gliozzi; Annalisa Relini; Michele Mazzanti; Carla Jodice (pp. 906-917).

Ataxin 1 (ATXN1) is the protein involved in spinocerebellar ataxia type 1, one of nine dominantly inherited neurodegenerative diseases triggered by polyglutamine expansion. One of the isolated polyglutamine tracts properties is to interact with lipid bilayers. Here we used a multidisciplinary approach to test whether one of the mechanisms responsible for neuronal degeneration involves the destabilization of the nuclear membrane. We thus analyzed the interaction between ATXN1 and lipid membranes, both on cellular models and on artificial lipid bilayers, comparing pathological expanded polyglutamine and histidine interrupted non-harmful polyglutamine tracts of the same length. The toxicity of the different constructs was tested in transiently transfected COS1 cells. Cells expressing pathological ATXN1 presented a significantly higher frequency of anomalous nuclei with respect to those expressing non-harmful ATXN1. Immunofluorescence and electron microscopy showed severe damage in the nuclear membrane of cells expressing the pathological protein. Atomic force microscopy on artificial membranes containing interrupted and non-interrupted partial ATXN1 peptides revealed a different arrangement of the peptides within the lipid bilayer. Force-distance measurements indicated that membrane fragility increases with the lengthening of the uninterrupted glutamine. Transmembrane electrical measurements were performed on artificial bilayers and on the inner nuclear membrane of ATXN1 full length transfected cells. Both artificial lipid bilayers and cellular models demonstrated the dynamic appearance of ionic pathways. Uninterrupted polyglutamines showed not only a larger ionic flow, but also an increase in the single event conductance. Collectively, our results suggest that expanded ATXN1 may induce unregulated ionic pathways in the nuclear membrane, causing severe damage to the cell.► One of the mechanisms causing SCA1 neurodegeneration could involve membrane destabilization. ► We analyzed the interaction between normal or expanded ataxin 1 and lipid membranes. ► Morphological analyses revealed nuclear membrane damage of cells expressing toxic ataxin 1. ► Atomic force microscopy and electrophysiology showed increased membrane fragility and permeability. ► We suggest that expanded ataxin 1 induces unregulated ionic pathways in the nuclear membrane.

Keywords: Abbreviations; SCA1; spinocerebellar ataxia type 1; ATXN1; ataxin 1; polyglutamine; polyQ; SBMA; spinal bulbar muscular atrophy; HD; Huntington disease; DRPLA; dentatorubropallidoluysian atrophy; NLS; nuclear localization signal; RBM17; RNA-binding motif protein 17; AFM; atomic force microscopeSpinocerebellar ataxia type 1; Ataxin 1; Polyglutamine disease; Neurodegeneration; Membrane; Unregulated ionic pathway

Downregulation of Metallothionein 1F, a putative oncosuppressor, by loss of heterozygosity in colon cancer tissue by Dong-Wang Yan; Jun-Wei Fan; Zhen-hai Yu; Ming-xue Li; Yu-Gang Wen; Da-Wei Li; Chong-Zhi Zhou; Xiao-Liang Wang; Quan Wang; Hua-Mei Tang; Zhi-Hai Peng (pp. 918-926).

Downregulation of metallothionein ( MT) genes has been reported in several tumors with discrepant results. This study is to investigate molecular mechanism of MT gene regulation in colon cancer which is characterized by tumor suppressor gene alterations.Integral analysis of microarray data with loss of heterozygosity (LOH) information was employed. Quantitative real-time PCR and immunohistochemistry were used to validate MT isoform expression in colon cancer tissues and cell lines. The effects of MT1F expression on RKO cell survival and tumorigenesis was analyzed. Bisulphite sequencing PCR (BSP) and methylation-specific PCR were employed to detect the methylation status of the MT1F gene in colon cancer tissues and cell lines. DNA sequencing was used to examine the LOH at the MT1F locus. MT1F, MT1G, MT1X, and MT2A gene expression was significantly downregulated in colon cancer tissue ( p<0.05). Exogenous MT1F expression increased RKO cell apoptosis and inhibited RKO cell migration, invasion and adhesion as well as in vivo tumorigenicity. Downregulation of MT1F gene in majority of human colon tumor tissues is mainly through mechanism by loss of heterozygosity ( p=0.001) while CpG island methylation of MT1F gene promoter region was only observed in poorly differentiated, MSI-positive RKO and LoVo colon cancer cell lines.MT1F is a putative tumor suppressor gene in colon carcinogenesis that is downregulated mainly by LOH in colon cancer tissue. Further studies are required to elucidate a possible role for MT1F downregulation in colon cancer initiation and/or progression.► The expression of MT1F, MT1G, MT1X, and MT2A was downregulated in colon cancer tissue. ► MT protein expression was negatively associated with tumor status. ► Exogenous MT1F expression increased RKO cell apoptosis. ► Exogenous MT1F expression inhibited in vivo tumorigenicity. ► Loss of heterozygosity is the main reason for MT1F mRNA downregulation in colon cancer tissues.

Keywords: Microarray; Metallothionein; Colon carcinogenesis; Epigenetics; Loss of heterozygosity

Effect of Tie-2 conditional deletion of BDNF on atherosclerosis in the ApoE null mutant mouse by Giuseppe Danilo Norata; Vivek Krishna Pulakazhi Venu; Elisa Callegari; Valentina Paloschi; Alberico Luigi Catapano (pp. 927-935).

The reduced expression (haplodeficiency) of the main brain derived neurotrophic factor receptor, namely TrkB is associated with reduced atherosclerosis, smooth muscle cells accumulation and collagen content in the lesion. These data support the concept that brain derived neurotrophic factor of vascular origin may contribute to atherosclerosis. However, to date, no experimental approach was possible to investigate this issue due to the lethality of brain derived neurotrophic factor null mice. To overcome these limitations, we generated a mouse model with a conditional deletion of brain derived neurotrophic factor in endothelial cells (Tie-2 Cre recombinase) on an atherosclerotic prone background (apolipoprotein E knock out) and investigated the effect of conditional brain derived neurotrophic factor deficiency on atherosclerosis. Despite brain derived neurotrophic factor reduction in the vascular wall, mice with conditional deletion of brain derived neurotrophic factor did not develop larger atherosclerotic lesion compared to controls. Smooth muscle cell content as well as the distribution of total and fibrillar collagen was similar in the atherosclerotic lesions from mice with brain derived neurotrophic factor conditional deficiency compared to controls. Finally an extended gene expression analysis failed to identify pro-atherogenic gene expression patterns among the animal with brain derived neurotrophic factor deficiency. In spite of the reduced brain derived neurotrophic factor expression, similar atherosclerosis development was observed in the brain derived neurotrophic factor conditional deficient mouse compared to controls. These pieces of evidence indicate that endothelial derived-brain derived neurotrophic factor is not a pro-atherogenic factor and would rather suggest to investigate the role of other TrkB activators on atherosclerosis.► The effect of endothelial BDNF deficiency on atherosclerosis was studied. ► In spite of the reduced BDNF expression, similar atherosclerosis development was observed. ► Endothelial derived-BDNF is not a pro-atherogenic factor. ► The role of other TrkB activators on atherosclerosis should be investigated.

Keywords: BNDF; Atherosclerosis; Vascular disorder

Overexpression of HIF-1α transgene in the renal medulla attenuated salt sensitive hypertension in Dahl S rats by Qing Zhu; Zhengchao Wang; Min Xia; Pin-Lan Li; Fan Zhang; Ningjun Li (pp. 936-941).

Hypoxia inducible factor (HIF)-1α-mediated gene activation in the renal medulla in response to high salt intake plays an important role in the control of salt sensitivity of blood pressure. High salt-induced activation of HIF-1α in the renal medulla is blunted in Dahl S rats. The present study determined whether the impairment of the renal medullary HIF-1α pathway was responsible for salt sensitive hypertension in Dahl S rats. Renal medullary HIF-1α levels were induced by either transfection of HIF-1α expression plasmid or chronic infusion of CoCl₂ into the renal medulla, which was accompanied by increased expressions of anti-hypertensive genes, cyclooxygenase-2 and heme oxygenase-1. Overexpression of HIF-1α transgenes in the renal medulla enhanced the pressure natriuresis, promoted the sodium excretion and reduced sodium retention after salt overload. As a result, hypertension induced by 2-week high salt was significantly attenuated in rats treated with HIF-1α plasmid or CoCl₂. These results suggest that an abnormal HIF-1α in the renal medulla may represent a novel mechanism mediating salt-sensitive hypertension in Dahl S rats and that induction of HIF-1α levels in the renal medulla could be a therapeutic approach for the treatment of salt-sensitive hypertension.► High salt-induced HIF-1α activation in the renal medulla is blunted in Dahl S rat. ► Induction of HIF-1α increased the expressions of anti-hypertensive genes. ► Induction of HIF-1α in the renal medulla enhanced sodium excretion. ► Induction of HIF-1α in the renal medulla attenuated salt-sensitive hypertension. ► Abnormal HIF-1α in the renal medulla is the cause for salt-sensitive hypertension.

Keywords: Pressure natriuresis; Heme oxygenase-1; Cyclooxygenase-2; Sodium excretion

Loss of c-Met accelerates development of liver fibrosis in response to CCl₄ exposure through deregulation of multiple molecular pathways by Jens U. Marquardt; Daekwan Seo; Luis E. Gómez-Quiroz; Koichi Uchida; Matthew C. Gillen; Mitsuteru Kitade; Pal Kaposi-Novak; Elizabeth A. Conner; Valentina M. Factor; Snorri S. Thorgeirsson (pp. 942-951).

HGF/c-Met signaling plays a pivotal role in hepatocyte survival and tissue remodeling during liver regeneration. HGF treatment accelerates resolution of fibrosis in experimental animal models. Here, we utilized Met^fl/fl;Alb-Cre^+/− conditional knockout mice and a carbon tetrachloride(CCl₄)-induced liver fibrosis model to formally address the role of c-Met signaling in hepatocytes in the context of chronic tissue injury. Histological changes during injury (4weeks) and healing phase (4weeks) were monitored by immunohistochemistry; expression levels of selected key fibrotic molecules were evaluated by western blotting, and time-dependent global transcriptomic changes were examined using a microarray platform. Loss of hepatocyte c-Met signaling altered hepatic microenvironment and aggravated hepatic fibrogenesis. Greater liver damage was associated with decreased hepatocyte proliferation, excessive stellate cell activation and rapid dystrophic calcification of necrotic areas. Global transcriptome analysis revealed a broad impact of c-Met on critical signaling pathways associated with fibrosis. Loss of hepatocyte c-Met caused a strong deregulation of chemotactic and inflammatory signaling ( MCP-1, RANTES, Cxcl10) in addition to modulation of genes involved in reorganization of the cytoskeletal network ( Actb, Tuba1a, Tuba8), intercellular communications and adhesion ( Adam8, Icam1, Itgb2), control of cell proliferation ( Ccng2, Csnk2a, Cdc6, cdk10), DNA damage and stress response ( Rad9, Rad52, Ercc4, Gsta1 and 2, Jun). Our study demonstrates that deletion of c-Met receptor in hepatocytes results in pronounced changes in hepatic metabolism and microenvironment, and establishes an essential role for c-Met in maintaining the structural integrity and adaptive plasticity of the liver under adverse conditions.► The molecular pathogenesis of CCl₄ –induced hepatic fibrogenesis. ► Specific loss of Met in hepatocytes increases hepatic fibrogenesis. ► Broad impact on inflammatory signaling, DNA damage and oxidative stress response. ► Disregulation of genes controlling structural integrity and cell proliferation. ► Met maintains the adaptive plasticity of the liver under adverse conditions.

Keywords: Abbreviations; HGF; hepatocyte growth factor; NAFLD/NASH; non-alcoholic fatty liver disease/steatohepatitis; ECM; extracellular matrix; MMP; matrix metalloproteinase; CCl; ₄; carbon tetrachloride; TGF-β; transforming growth factor β; PDGF-β; platelet derived growth factor β; BrdU; bromodeoxyuridine; IHC; immunohistochemistry; FDR; false discovery rate; HSC; hepatic stellate cells; GPCR; G-protein-coupled receptorc-met; Fibrosis; Microarray; CCl; ₄

The novel endocrine disruptor tolylfluanid impairs insulin signaling in primary rodent and human adipocytes through a reduction in insulin receptor substrate-1 levels by Robert M. Sargis; Brian A. Neel; Clifton O. Brock; Yuxi Lin; Allison T. Hickey; Daniel A. Carlton; Matthew J. Brady (pp. 952-960).

Emerging data suggest that environmental endocrine disrupting chemicals may contribute to the pathophysiology of obesity and diabetes. In a prior work, the phenylsulfamide fungicide tolylfluanid (TF) was shown to augment adipocyte differentiation, yet its effects on mature adipocyte metabolism remain unknown. Because of the central role of adipose tissue in global energy regulation, the present study tested the hypothesis that TF modulates insulin action in primary rodent and human adipocytes. Alterations in insulin signaling in primary mammalian adipocytes were determined by the phosphorylation of Akt, a critical insulin signaling intermediate. Treatment of primary murine adipose tissue in vitro with 100nM TF for 48h markedly attenuated acute insulin-stimulated Akt phosphorylation in a strain- and species-independent fashion. Perigonadal, perirenal, and mesenteric fat were all sensitive to TF-induced insulin resistance. A similar TF-induced reduction in insulin-stimulated Akt phosphorylation was observed in primary human subcutaneous adipose tissue. TF treatment led to a potent and specific reduction in insulin receptor substrate-1 (IRS-1) mRNA and protein levels, a key upstream mediator of insulin's diverse metabolic effects. In contrast, insulin receptor-β, phosphatidylinositol 3-kinase, and Akt expression were unchanged, indicating a specific abrogation of insulin signaling. Additionally, TF-treated adipocytes exhibited altered endocrine function with a reduction in both basal and insulin-stimulated leptin secretion. These studies demonstrate that TF induces cellular insulin resistance in primary murine and human adipocytes through a reduction of IRS-1 expression and protein stability, raising concern about the potential for this fungicide to disrupt metabolism and thereby contribute to the pathogenesis of diabetes.► Insulin signaling in primary rodent and human adipocytes was impaired by the fungicide tolylfluanid. ► Tolylfluanid specifically reduced the mRNA and protein expression of insulin receptor substrate-1. ► Reduction of insulin signaling resulted in decreased leptin release from primary adipocytes.

Keywords: Adipocyte; Insulin resistance; Environmental endocrine disruption

Calmodulin antagonizes amyloid-β peptides-mediated inhibition of brain plasma membrane Ca²⁺-ATPase by Maria Berrocal; M. Rosario Sepulveda; Maria Vazquez-Hernandez; Ana M. Mata (pp. 961-969).

The synaptosomal plasma membrane Ca²⁺-ATPase (PMCA) plays an essential role in regulating intracellular Ca²⁺ concentration in brain. We have recently found that PMCA is the only Ca²⁺ pump in brain which is inhibited by amyloid-β peptide (Aβ), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aβ. Results from kinetic assays indicate that Aβ aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aβ could be blocked by pretreating the purified protein with Ca²⁺-calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aβ. Dot-overlay experiments indicated a physical association of Aβ with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aβ by burying exposed sites on PMCA, making them inaccessible to Aβ, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca²⁺ dysregulation by PMCA inhibition induced by Aβ.► Neurotoxic Aβ peptide inhibits synaptosomal PMCA in an aggregation-dependent manner. ► Modulation of PMCA by calmodulin can provide neuroprotection against Aβ toxicity. ► Calmodulin may play a key role in restoring Ca²⁺ dysregulation in Alzheimer's disease.

Keywords: Abbreviations; Aβ; amyloid-β peptide; AD; Alzheimer's disease; PBS; Phosphate buffered saline; PC; Phosphatidylcholine; PMCA; Plasma membrane Ca; ²⁺; -ATPase; TBS; Tris buffered salineAmyloid β-peptide; PMCA; Calmodulin; Chymotrypsin; Calpain

Protein profiling reveals energy metabolism and cytoskeletal protein alterations in LMNA mutation carriers by Cinzia Magagnotti; Angela Bachi; Gianpaolo Zerbini; Elena Fattore; Isabella Fermo; Michela Riba; Stefano C. Previtali; Maurizio Ferrari; Annapaola Andolfo; Sara Benedetti (pp. 970-979).

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery–Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.► This work is the first proteomic study on ex-vivo cells from LMNA mutant patients. ► Cytoskeletal/structural proteins are down-regulated in LMNA mutant patients. ► Major alterations are reflected into energy production. ► LMNA mutations alter the oxidative stress response.

Keywords: Laminopathies; Mass spectrometry; Muscular dystrophy; Proteomics; Skin fibroblasts

Sustained intrahepatic glutathione depletion causes proteasomal degradation of multidrug resistance-associated protein 2 in rat liver by Shuichi Sekine; Kaori Mitsuki; Kousei Ito; Satoe Kugioka; Toshiharu Horie (pp. 980-987).

Multidrug resistance-associated protein 2 (MRP2) is a member of a family of efflux transporters that are involved in biliary excretion of organic anions from hepatocytes. Disrupted canalicular localization and decreased protein expression of MRP2 have been observed in patients with chronic cholestatic disorder and hepatic failure without a change in its mRNA expression. We have previously demonstrated that post-transcriptional regulation of the rapid retrieval of rat MRP2 from the canalicular membrane to the intracelluar compartment occurs under conditions of acute (~30min) oxidative stress. However, it is unclear whether MRP2 expression is decreased during its sustained internalization during chronic oxidative stress. The present study employed buthionine sulfoximine (BSO) to induce chronic oxidative stress in the livers of Sprague–Dawley rats and then examined the protein expression and localization of MRP2. Canalicular MRP2 localization was altered by BSO treatment for 2h without changing the hepatic protein expression of MRP2. While the 8h after exposure to BSO, hepatic MRP2 protein expression was decreased, and the canalicular localization of MRP2 was disrupted without changing the mRNA expression of MRP2. The BSO-induced reduction in MRP2 protein expression was suppressed by pretreatment with N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal ( MG-132), a proteasomal inhibitor. Furthermore, the modification of MRP2 by small ubiquitin-relatedmodifier 1 (SUMO-1) was impaired in BSO-treated rat liver,while that by ubiquitin (Ub) and MRP2 was enhanced. Taken together, the results of this study suggest the sustained periods of low GSH content coupled with altered modification of MRP2 by Ub/SUMO-1 were accompanied by proteasomal degradation of MRP2.► At 2h after BSO treatment, intrahepatic GSH decrease causes Mrp2 internalization. ► At 8h after BSO treatment, Mrp2 protein expression is decreased in proteasomal-degradation dependent manner. ► Ubiquitination and de-SUMOylation of Mrp2 can be observed in the liver treated with BSO for 8h.

Keywords: Abbreviations; ARC; apoptosis repressor with caspase recruitment domain; BSA; bovine serum albumin; Bsep/BSEP; bile salt export pump; BSO; buthionine-sulfoximine; CD26; cluster of differentiation 26; CrM; crude membrane; DJS; Dubin-Johnson syndrome; EA; ethacrynic acid; GADPH; glyceraldehyde-3-phosphate dehydrogenase; γ-GCS; γ-glutamyl cysteine synthetase; GSH; glutathione; HPLC; high performance liquid chromatography; LPS; lipopolysaccharide; MRP2; multidrug resistance-associated protein 2; PFIC; progressive familial intrahepatic cholestasis; PDZK1; PSD95/Dlg/ZO1-containing 1; PKC; protein kinase C; SUMO; small ubiquitin-like modifier; t-BHP; tert-butyl hydroperoxide; TTBS; Tween 20/Tris-buffered saline; Ub; ubiquitin; UBA2; ubiquitin-activating enzyme 2; Ubc9; ubiquitin-conjugating enzyme 9Oxidative stress; MRP2; Degradation; SUMO; Ubiquitin; Proteasome

Possible roles of DLK1 in the Notch pathway during development and disease by Farah A. Falix; Daniël C. Aronson; Wouter H. Lamers; Ingrid C. Gaemers (pp. 988-995).

The Delta-Notch pathway is an evolutionarily conserved signaling pathway which controls a broad range of developmental processes including cell fate determination, terminal differentiation and proliferation. In mammals, four Notch receptors (NOTCH1–4) and five activating canonical ligands (JAGGED1, JAGGED2, DLL1, DLL3 and DLL4) have been described. The precise function of noncanonical Notch ligands remains unclear. Delta-like 1 homolog (DLK1), the best studied noncanonical Notch ligand, has been shown to act as an inhibitor of Notch signaling in vitro, but its function in vivo is poorly understood. In this review we summarize Notch signaling during development and highlight recent studies in DLK1expression that reveal new insights into its function.► DLK1 is involved in branching morphogenesis and terminal differentiation. ► DLK1 is a modulator of Notch signaling. ► DLK1 in pediatric tumors indicates developmental arrest of specific cell types.

Keywords: DLK1; Notch pathway; Development; Adipose tissue; Liver; Pediatric tumor

Overexpression of insulin like growth factor binding protein 5 reduces liver fibrosis in chronic cholangiopathy by Sokolovic Aleksandar Sokolović; Paula S. Montenegro-Miranda; Dirk Rudi de Waart; Radha M.N. Cappai; Suzanne Duijst; Sokolovic Milka Sokolović; Piter J. Bosma (pp. 996-1003).

The ATP-binding cassette, sub-family B member 4 knock-out mouse (Abcb4^−/−) is a relevant model for chronic cholangiopathy in man. Due to the lack of this P-glycoprotein in the canalicular membrane of hepatocytes, the secretion of phospholipids into bile is absent, resulting in increased bile toxicity. Expression of insulin like growth factor binding protein 5 ( Igfbp5) increases in time in the livers of these mice. It is unclear whether this induction is a consequence of or plays a role in the progression of liver pathology. The aim of this study was therefore to investigate the effect of IGFBP5 induction on the progression of liver fibrosis caused by chronic cholangiopathy. IGFBP5 and, as a control, green fluorescent protein were overexpressed in the hepatocytes of Abcb4^−/− mice, using an adeno-associated viral vector (AAV). Progression of liver fibrosis was studied 3, 6, and 12weeks after vector injection by analyzing serum parameters, collagen deposition, expression of pro-fibrotic genes, inflammation and oxidative stress. A single administration of the AAV vectors provided prolonged expression of IGFBP5 and GFP in the livers of Abcb4^−/− mice. Compared to GFP control, fractional liver weight, extracellular matrix deposition and amount of activated hepatic stellate cells significantly decreased in IGFBP5 overexpressing mice even 12weeks after treatment. This effect was not due to a change in bile composition, but driven by reduced inflammation, oxidative stress, and proliferation. Overexpression of IGFBP5 seems to have a protective effect on liver pathology in this model for chronic cholangiopathy.► IGFBP5 was overexpressed in the livers of Abcb4^−/− mice using AAV-delivery. ► ECM deposition significantly decreased with IGFBP5 overexpression. ► Hepatocyte proliferation, inflammation and oxidative stress were reduced by IGFBP5. ► Overexpression of IGFBP5 reduces liver fibrosis caused by chronic cholangiopathy.

Keywords: Abbreviations; AAV; adeno-associated virus; Abcb4; ^−/−; ATP-binding cassette, sub-family B member 4; ECM; extracellular matrix; F4/80; EGF-like module containing, mucin-like, hormone receptor-like sequence 1; GFP; green fluorescent protein; GSH; glutathione; GSSG; glutathione disulfide; HSC; hepatic stellate cells; IGFBP5; insulin-like growth factor-binding protein 5; Mac1; integrin alpha M; MFs; myofibroblasts; MPO; myeloperoxidase; p21; cyclin-dependent kinase inhibitor 1A; PCNA; proliferating cell nuclear antigen; α-SMA; actin, alpha 2, smooth muscleliver fibrosis; Abcb4; ^−/−; mice; IGFBP5; AAV; inflammation; systems genetics

Anti-proliferative effect of atrial natriuretic peptide on colorectal cancer cells: Evidence for an Akt-mediated cross-talk between NHE-1 activity and Wnt/β-catenin signaling by Annalucia Serafino; Noemi Moroni; Rossana Psaila; Manuela Zonfrillo; Federica Andreola; Francesca Wannenes; Luana Mercuri; Guido Rasi; Pasquale Pierimarchi (pp. 1004-1018).

Acidic tumor microenvironment and Wnt/β-catenin pathway activation have been recognized as two crucial events associated with the initiation and progression of cancer. The aim of this study was to clarify the molecular mechanisms underlying the anti-proliferative effects of atrial natriuretic peptide (ANP) as well as to investigate the relationship between the cellular pH and the Wnt/β-catenin signaling in cancer cells.To pursue our aims, we conducted investigations in DHD/K12/Trb rat colon adenocarcinoma cells. Intracellular pH was measured by Confocal Laser Scanning Microscopy (CLSM) using the lysosensor Green DND-189 probe. Expression of crucial molecules in the Wnt/β-catenin signaling pathway was analyzed by CLSM, western blot, and real time PCR. Measurements of activation (phosphorylation state) of Akt, ERK1/2, and p38MAPKinase were performed by Reverse-Phase Protein Microarray Analysis (RPMA).We showed that ANP triggered a NHE-1-mediated increase of the intracellular acidity, inhibiting the Wnt/β-catenin signaling simultaneously. Moreover, we observed that the Wnt1a, a Wnt signaling activator, affected the intracellular pH in an opposite fashion. Results from the comparative analysis of ANP and EIPA (a NHE-1 specific inhibitor) showed that these two molecules affect both the intracellular acidification and the Wnt/β-catenin signaling cascade. Specifically, ANP acts on the upstream of the cascade, through a Frizzled-mediated activation, while EIPA does on the downstream.We show for the first time that the Akt activity might be a relevant molecular event linking the NHE-1-regulated intracellular pH and the Wnt/β-catenin signaling. This provides evidence for a cross-talk between the intracellular alkalinization and the Wnt signaling in tumor cells.Display Omitted► ANP increases the intracellular acidity and inhibits the Wnt signaling in cancer cells. ► The Wnt signaling activator Wnt1a affected the intracellular pH in an opposite fashion. ► ANP affects cellular pH and Wnt signaling by a Frizzled receptor-mediated mechanism. ► Akt activity interconnects the NHE-1-regulated pH and the Wnt signaling in cancer cells.

Keywords: Abbreviations; ANP; atrial natriuretic peptide; APC; adenomatous polyposis coli; CLSM; confocal laser scanning microscopy; EIPA; 5-(Nethyl-N-isopropyl)amiloride; GSK-3β; glycogen synthase kinase 3β; NHE; Sodium/Proton Exchanger; QPCR; Quantitative Reverse Transcription Polymerase Chain Reaction; RPMA; Reverse-Phase Protein Microarray AnalysisColon adenocarcinoma cell; NHE-1 inhibition; Tumor microenvironmental pH; Wnt pathway; Akt

Metabolically induced heteroplasmy shifting andl-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS by Valerie Desquiret-Dumas; Naig Gueguen; Magalie Barth; Arnaud Chevrollier; Saege Hancock; Douglas C. Wallace; Patrizia Amati-Bonneau; Daniel Henrion; Dominique Bonneau; Pascal Reynier; Vincent Procaccio (pp. 1019-1029).

The m.3243A>G variant in the mitochondrial tRNA^Leu(UUR) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood.We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition ofl-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition ofl-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting andl-arginine therapy may constitute promising therapeutic strategies against MELAS.► The m.3243A>G mutation induces a metabolic switch in SH-SY5Y cybrid cells. ► Oxidative stress contributes to the pathophysiology of MELAS. ► MELAS neuronal-like cells are a good model for the evaluation of therapeutic strategies. ► Heteroplasmy shifting can alleviate mitochondrial respiratory chain dysfunction. ►l-arginine, a nitric oxide (NO) donor, reduces complex I activity in a MELAS neuronal-like model.

Keywords: Abbreviations; CNS; central nervous system; COX; cytochrome; c; oxidase; FBS; fetal bovine serum; GSSG; glutathione disulfide; GSH; glutathione; LDH; lactate dehydrogenase; MnSOD; manganese superoxide dismutase; mtDNA; mitochondrial DNA; MELAS; mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; NO; nitric oxide; RFLP; restriction fragment length polymorphism; ROS; reactive oxygen speciesMitochondria; Mitochondrial disorder; mtDNA; MELAS; Neuronal cell; l; -arginine

Focal adhesion kinase negatively regulates neuronal insulin resistance by Amit Gupta; Bharti Bisht; Chinmoy Sankar Dey (pp. 1030-1037).

Focal adhesion kinase (FAK), a non-receptor protein kinase, is known to be a phosphatidyl inositol 3-kinase (PI3K) pathway activator and thus widely implicated in regulation of cell survival and cancer. In recent years FAK has also been strongly implicated as a crucial regulator of insulin resistance in peripheral tissues like skeletal muscle and liver, where decrease in its expression/activity has been shown to lead to insulin resistance. However, in the present study we report an altogether different role of FAK in regulation of insulin/PI3K signaling in neurons, the post-mitotic cells. An aberrant increase in FAK tyrosine phosphorylation was observed in insulin resistant Neuro-2a (N2A) cells. Downregulation of FAK expression utilizing RNAi mediated gene silencing in insulin resistant N2A cells completely ameliorated the impaired insulin/PI3K signaling and glucose uptake. FAK silencing in primary cortical neurons also showed marked enhancement in glucose uptake. The results thus suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. Interestingly, the available literature also demonstrates cell-type specific functions of FAK in neurons. FAK that is well known for its cell survival effects has been shown to be involved in neurodegeneration. Along with these previous reports, present findings highlight a novel and critical role of FAK in neurons. Moreover, as this implicates differential regulation of insulin/PI3K pathway by FAK in peripheral tissues and neuronal cells, it strongly suggests precaution while considering FAK modulators as possible therapeutics.► FAK is known to be a positive regulator of insulin/PI3K signaling in peripheral tissues. ► Aberrant activation of FAK was observed under neuronal insulin resistance. ► Silencing FAK completely ameliorated neuronal insulin resistance. ► Results suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. ► Findings highlight a novel and critical role of FAK in neurons, the post-mitotic cells.

Keywords: FAK; Insulin resistance; Neuronal cells

Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle by Corey J. Miller; Sellamuthu S. Gounder; Sankaranarayanan Kannan; Karan Goutam; Vasanthi R. Muthusamy; Matthew A. Firpo; J. David Symons; Robert Paine III; John R. Hoidal; Namakkal Soorappan Rajasekaran (pp. 1038-1050).

Age-associated decline in antioxidant potential and accumulation of reactive oxygen/nitrogen species are primary causes for multiple health problems, including muscular dystrophy and sarcopenia. The role of the nuclear erythroid-2-p45-related factor-2 (Nrf2) signaling has been implicated in antioxidant gene regulation. Here, we investigated the loss-of-function mechanisms for age-dependent regulation of Nrf2/ARE (Antioxidant Response Element) signaling in skeletal muscle (SM). Under basal physiological conditions, disruption of Nrf2 showed minimal effects on antioxidant defenses in young (2months) Nrf2−/− mice. Interestingly, mRNA and protein levels of NADH Quinone Oxidase-1 were dramatically (* P<0.001) decreased in Nrf2−/− SM when compared to WT at 2months of age, suggesting central regulation of NQO1 occurs through Nrf2. Subsequent analysis of the Nrf2-dependent transcription and translation showed that the aged mice (>24months) had a significant increase in ROS along with a decrease in glutathione (GSH) levels and impaired antioxidants in Nrf2−/− when compared to WT SM. Further, disruption of Nrf2 appears to induce oxidative stress (increased ROS, HNE-positive proteins), ubiquitination and pro-apoptotic signals in the aged SM of Nrf2−/− mice. These results indicate a direct role for Nrf2/ARE signaling on impairment of antioxidants, which contribute to muscle degradation pathways upon aging. Our findings conclude that though the loss of Nrf2 is not amenable at younger age; it could severely affect the SM defenses upon aging. Thus, Nrf2 signaling might be a potential therapeutic target to protect the SM from age-dependent accumulation of ROS by rescuing redox homeostasis to prevent age-related muscle disorders such as sarcopenia and myopathy.► Nrf2-Keap1 pathway is conserved in skeletal muscle. ► Abrogation of Nrf2 has a minimal role on SM redox state in younger age. ► Loss of Nrf2 down regulates transcription of ARE-antioxidants upon aging. ► Impaired antioxidant mechanisms induce oxidative stress in aged Nrf2−/− mice. ► Disruption of Nrf2 increases ubiquitination and apoptosis upon aging.

Keywords: Nrf2; ARE-signaling; ROS; EPR; Ubiquitination; Aging

Calorie restriction prevents the development of insulin resistance and impaired insulin signaling in skeletal muscle of ovariectomized rats by Mujalin Prasannarong; Kanokwan Vichaiwong; Vitoon Saengsirisuwan (pp. 1051-1061).

Insulin resistance of skeletal muscle glucose transport due to prolonged loss of ovarian function in ovariectomized (OVX) rats is accompanied by other features of the metabolic syndrome and may be confounded by increased calorie consumption. In this study, we investigated the role of calorie consumption in the development of insulin resistance in OVX rats. In addition, we examined the cellular mechanisms underlying skeletal muscle insulin resistance in OVX rats. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). OVX rats either had free access to food, pair feeding (PF) with SHAM or received a 35% reduction in food intake (calorie restriction; CR) for 12weeks. Compared with SHAM, ovariectomy induced skeletal muscle insulin resistance, which was associated with decreases (32–70%) in tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), IRS-1 associated p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and Akt Ser⁴⁷³ phosphorylation whereas insulin-stimulated phosphorylation of IRS-1 Ser³⁰⁷, SAPK/JNK Thr¹⁸³/Tyr¹⁸⁵, and p38 mitogen-activated protein kinase (MAPK) Thr¹⁸⁰/Tyr¹⁸² was increased (24–62%). PF improved the serum lipid profile but did not restore insulin-stimulated glucose transport, indicating that insulin resistance in OVX rats is a consequence of ovarian hormone deprivation. In contrast, impaired insulin sensitivity and defective insulin signaling were not observed in the skeletal muscle of OVX+CR rats. Therefore, we provide evidence for the first time that CR effectively prevents the development of insulin resistance and impaired insulin signaling in the skeletal muscle of OVX rats.►Insulin resistance in OVX is caused by ovarian hormone deprivation, not over-feeding. ►Impaired insulin signaling and enhanced MAPK are observed in skeletal muscle of OVX. ►Elevated MAPK is not required for insulin resistance in skeletal muscle of OVX. ►CR improves glucose tolerance and insulin signaling in skeletal muscle of OVX rats.

Keywords: Insulin resistance; Calorie restriction; Skeletal muscle; Insulin signaling; MAPK; Ovariectomy

A constant and similar assembly defect of mitochondrial respiratory chain complex I allows rapid identification of NDUFS4 mutations in patients with Leigh syndrome by Z. Assouline; M. Jambou; M. Rio; C. Bole-Feysot; P. de Lonlay; C. Barnerias; I. Desguerre; C. Bonnemains; C. Guillermet; J. Steffann; A. Munnich; J.P. Bonnefont; Rotig A. Rötig; A.S. Lebre (pp. 1062-1069).

Isolated complex I deficiency is a frequent cause of respiratory chain defects in childhood. In this study, we report our systematic approach with blue native PAGE (BN-PAGE) to study mitochondrial respiratory chain assembly in skin fibroblasts from patients with Leigh syndrome and CI deficiency. We describe five new NDUFS4 patients with a similar and constant abnormal BN-PAGE profile and present a meta-analysis of the literature. All NDUFS4 mutations that have been tested with BN-PAGE result in a constant and similar abnormal assembly profile with a complete loss of the fully assembled complex I usually due to a truncated protein and the loss of its canonical cAMP dependent protein kinase phosphorylation consensus site. We also report the association of abnormal brain MRI images with this characteristic BN-PAGE profile as the hallmarks of NDUFS4 mutations and the first founder NDUFS4 mutations in the North-African population.► Blue native PAGE to study respiratory chain assembly in patients with complex I deficiency ► Description of five new NDUFS4 patients identified after BN-PAGE study ► Constant and similar abnormal complex I assembly profile in all known NDUFS4 mutations ► Association of abnormal brain MRI images and BN-PAGE profile as the hallmarks of NDUFS4 mutations ► First founder NDUFS4 mutations in the North-African population.

Keywords: Abbreviations; CI; complex I; BN-PAGE; blue native-polyacrylamide gel electrophoresis; MRI; Magnetic Resonance Imaging; mtDNA; mitochondrial DNA, RC, respiratory chain; kDa; kilodaltons; OXPHOS; oxidative phosphorylation enzymes; PND; prenatal diagnosisMitochondrial disorder; Respiratory chain complex I assembly; BN-PAGE; NDUFS4; gene; Leigh syndrome; Founder mutation

Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures by Tobias Jochum; Manuela E. Ritz; Christoph Schuster; Sarah F. Funderburk; Katja Jehle; Katja Schmitz; Falko Brinkmann; Michael Hirtz; David Moss; Andrew C.B. Cato (pp. 1070-1078).

Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300–600nm in length. These were clearly different from annular oligomers 120–180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.► AR oligomeric structures exists in two forms, a fibrillar toxic form and an annular non-toxic form. ► This explains the fact that AR aggregates occur in healthy individuals with no polyQ-extension. ► Specific mutations outside the polyQ stretch of the wild-type AR induce fibrillar aggregation. ► The SBMA modifier melatonin blocks toxic fibrillar and induces non-toxic annular aggregates.

Keywords: Abbreviations; AFM; atomic force microscopy; AR; androgen receptor; DHT; dihydrotestosterone; polyQ; polyglutamine; SBMA; Spinal and Bulbar Muscular AtrophySBMA; Aggregation; Polyglutamine; Androgen receptor; Melatonin; Neurodegeneration

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BBA - Molecular Basis of Disease (v.1822, #6)