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BBA - Molecular Basis of Disease (v.1812, #4)

Editorial Board (pp. i).

Abnormal metabolism flexibility in response to high palmitate concentrations in myotubes derived from obese type 2 diabetic patients by Magali Kitzmann; Louise Lantier; Hebrard Sophie Hébrard; Jacques Mercier; Marc Foretz; Celine Aguer (pp. 423-430).

Insulin resistance in type 2 diabetes (T2D) is associated with intramuscular lipid (IMCL) accumulation. To determine whether impaired lipid oxidation is involved in IMCL accumulation, we measured expression of genes involved in mitochondrial oxidative metabolism or biogenesis, mitochondrial content and palmitate beta-oxidation before and after palmitate overload (600μM for 16h), in myotubes derived from healthy subjects and obese T2D patients. Mitochondrial gene expression, content and network were not different between groups. Basal palmitate beta-oxidation was not affected in T2D myotubes, whereas after 16h of palmitate pre-treatment, T2D myotubes in contrast to control myotubes, showed an inability to increase palmitate beta-oxidation ( p<0.05). Interestingly, acetyl-CoA carboxylase (ACC) phosphorylation was increased with a tendency for statistical significance after palmitate pre-treatment in control myotubes ( p=0.06) but not in T2D myotubes which can explain their inability to increase palmitate beta-oxidation after palmitate overload. To determine whether the activation of the AMP activated protein kinase (AMPK)-ACC pathway was able to decrease lipid content in T2D myotubes, cells were treated with AICAR and metformin. These AMPK activators had no effect on ACC and AMPK phosphorylation in T2D myotubes as well as on lipid content, whereas AICAR, but not metformin, increased AMPK phosphorylation in control myotubes. Interestingly, metformin treatment and mitochondrial inhibition by antimycin induced increased lipid content in control myotubes. We conclude that T2D myotubes display an impaired capacity to respond to metabolic stimuli.► Mitochondrial content is normal in type 2 diabetic myotubes ► Palmitate oxidation is decreased in type 2 diabetic myotubes compared to control myotubes only in condition of lipid overload ► ACC inactivation by palmitate is impaired in type 2 diabetic myotubes ► AICAR and metformin treatments do not activate AMPK or decrease lipid content in type 2 diabetic myotubes ► Mitochondrial inhibition by antimycin or metformin induce an increase in lipid content in control myotubes.

Keywords: Abbreviations; T2D; type 2 diabetes; IMCL; intramuscular lipid; ACC; acetyl-CoA carboxylase; AMPK; AMP activated protein kinase; AICAR; AMP-mimetic 5-aminoimidazole-4-carboxamide-1-β-; d; -ribofuranoside; FA; fatty-acid; ASM; acid-soluble metabolites; CS; citrate synthase; HAD; 3-hydroxy-acyl-CoA-dehydrogenase; NRF1; nuclear respiratory factor 1; CPT1; carnitine palmitoyltransferase 1Lipid overflow; Palmitate beta-oxidation; Mitochondria; Acetyl-CoA carboxylase; AMP activated protein kinase

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity by L. Ulianich; G. Terrazzano; M. Annunziatella; G. Ruggiero; F. Beguinot; B. Di Jeso (pp. 431-438).

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.► ER stress impairs MHC-I surface expression in thyroid cells. ► Thyroid cells undergoing ER stress are more susceptible to NK-mediated lysis. ► Thyroid cells undergoing ER stress stimulate INF-γ production by NK cells. ► These results suggest a possible new role of ER stress and NK activation in thyroid autoimmunity.

Keywords: Abbreviations; BiP; binding protein (GRP78); ER; endoplasmic reticulum; ERAD; ER associated degradation; GRP94; glucose regulated protein 94; HFE; hereditary hemochromatosis protein; HLA-I; human leukocyte antigen-I; IFN-γ; interferon-γ; IL-2; interleukin-2; MHC-I; major histocompatibility complex class I; NK; natural killer; PDI; protein disulfide isomerase; TH; thapsigargin; TN; tunicamycin; UPR; unfolded protein response; XBP-1; X-box binding protein 1ER stress; Natural killer cells; MHC-I expression; Thyroid autoimmunity

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy by Marni D. Boppart; Dean J. Burkin; Stephen J. Kaufman (pp. 439-446).

Transgenic expression of the α7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy ( mdx/utr^−/−) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the α7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remains to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by α7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr^−/− and α7BX2- mdx/utr^−/− mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr^−/− muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr^−/− mice overexpressing the α7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in α7BX2- mdx/utr^−/− compared to mdx/utr^−/− mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL⁺ cells observed in α7BX2- mdx/utr^−/− mice. We conclude that the α7β1 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.►Signaling is altered in a mouse model of Duchenne muscular dystrophy ( mdx/utr^−/−). ►AKT signaling may provide the mechanistic basis for α7 integrin-mediated therapy. ►ILK associates with α7B integrin at a critical tyrosine residue upon activation.

Keywords: α7 Integrin; Muscular dystrophy; Integrin linked kinase (ILK); AKT; Cell signaling; Apoptosis

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy by Marni D. Boppart; Dean J. Burkin; Stephen J. Kaufman (pp. 439-446).

Keywords: α7 Integrin; Muscular dystrophy; Integrin linked kinase (ILK); AKT; Cell signaling; Apoptosis

Lipotoxicity and steatohepatitis in an overfed mouse model for non-alcoholic fatty liver disease by Ingrid C. Gaemers; Jan M. Stallen; Cindy Kunne; Christian Wallner; Jochem van Werven; Aart Nederveen; Wouter H. Lamers (pp. 447-458).

The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH.Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied.Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ ( Pparγ), fibroblast growth factor 21 ( Fgf21), fatty acid binding protein ( Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice.In conclusion: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.►The consecutive stages of NAFLD can be recreated in time in an overfed mouse model. ►Pparγ and Fgf21 are induced in the livers of HFLD-overfed mice only. ►Steatohepatitis coincides with obesity, hyperinsulinemia and loss of liver glycogen. ►Steatohepatitis coincides with the occurrence of hepatic endoplasmatic reticulum stress. ►Adipose tissue of overfed mice is inflamed and has reduced adiponectin expression.

Keywords: Non-alcoholic fatty liver disease; Steatohepatitis; Liver; Overnutrition; High-fat diet; Endoplasmatic reticulum stress; Adipose tissue

Peripheral analgesia: Hitting pain where it hurts by Jason J. McDougall (pp. 459-467).

Pain is a complex biological phenomenon that encompasses intricate neurophysiological, behavioural, psychosocial and affective components. Protracted or chronic pain alerts an individual to a possible pathological abnormality and is the main reason why patients visit a primary care physician. Despite the pervasiveness of chronic pain in the population, the effectiveness of current pharmacological therapies remains woefully inadequate and prolonged treatment often leads to the development of undesirable side-effects. Since the vast majority of chronic pain originates in a specific tissue or group of tissues, it may be advantageous to target pain control in the periphery and thereby circumvent the known risks associated with non-specific systemic treatments. This review spotlights a number of promising targets for peripheral pain control including the transient receptor potential (TRP) family of neuronal ion channels, the family of proteinase activated receptors (PARs), cannabinoids, and opioids. A critical appraisal of these targets in preclinical models of disease is given and their suitability as future peripheral analgesics is discussed.► An appraisal of peripheral analgesics in preclinical animal models of disease is given. ► Highlights the role of transient receptor potential (TRP) ion channels. ► Highlights the role of proteinase activated receptors (PARs). ► Highlights the role of cannabinoids. ► Highlights the role of opioids.

Keywords: Animal model of disease; Nociception; Novel target; Pain; Peripheral analgesia

Peripheral analgesia: Hitting pain where it hurts by Jason J. McDougall (pp. 459-467).

Keywords: Animal model of disease; Nociception; Novel target; Pain; Peripheral analgesia

Point mutated caveolin-3 form (P104L) impairs myoblast differentiation via Akt and p38 signalling reduction, leading to an immature cell signature by Elena Stoppani; Stefania Rossi; Elisabetta Meacci; Fabio Penna; Paola Costelli; Arianna Bellucci; Fiorella Faggi; Daniele Maiolo; Eugenio Monti; Alessandro Fanzani (pp. 468-479).

Unbalanced levels of caveolin-3 (Cav3) are involved in muscular disorders. In the present study we show that differentiation of immortalized myoblasts is affected by either lack or overexpression of Cav3. Nevertheless, depletion of Cav3 induced by delivery of the dominant-negative Cav3 (P104L) form elicited a more severe phenotype, characterized by the simultaneous attenuation of the Akt and p38 signalling networks, leading to an immature cell and molecular signature. Accordingly, differentiation of myoblasts harbouring Cav3 (P104L) was improved by countering the reduced Akt and p38 signalling network via administration of IGF-1 or trichostatin A. Furthermore, loss of Cav3 correlated with a deregulation of the TGF-β-induced Smad2 and Erk1/2 pathways, confirming that Cav3 controls TGF-β signalling at the plasma membrane. Overall, these data suggest that loss of Cav3, primarily causing attenuation of both Akt and p38 pathways, contributes to impair myoblast fusion.► Cav3 loss of expression triggered by Cav3 (P104L) form impairs Akt and p38 signalling in myoblasts. ► Cav3 (P104L) myoblasts display an immature cell and molecular signature. ► Loss of Cav3 at the plasma membrane perturbs TGF-beta pathways. ► Administration of IGF-1 or trichostatin A improves the differentiation of myoblasts harbouring Cav3 (P104L) form.

Keywords: Abbreviations; bHLH; basic helix-loop-helix; BSA; bovine serum albumin; CMV; cytomegalovirus; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethylsulfoxide; FBS; foetal bovine serum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; HS; horse serum; IGF-1; Insulin-like growth factor-1; MAPK; mitogen-activated protein kinases; MCK; muscle creatine kinase; MLC; myosin light chain; MMLV-RT; moloney murine leukemia virus reverse transcriptase; MyHC; myosin heavy chain; PBS; phosphate buffer solution; PI3K; phosphatydil-inositol-3-kinase; TGF-β; transforming growth factors betaCaveolin-3; Skeletal myoblasts; Akt; p38 MAPK; TGF-β

Biliverdin reductase-A protein levels and activity in the brains of subjects with Alzheimer disease and mild cognitive impairment by Eugenio Barone; Fabio Di Domenico; Giovanna Cenini; Rukhsana Sultana; Chiara Cini; Paolo Preziosi; Marzia Perluigi; Cesare Mancuso; D. Allan Butterfield (pp. 480-487).

Biliverdin reductase-A is a pleiotropic enzyme involved not only in the reduction of biliverdin-IX-alpha into bilirubin-IX-alpha, but also in the regulation of glucose metabolism and cell growth secondary to its serine/threonine/tyrosine kinase activity. Together with heme oxygenase, whose metabolic role is to degrade heme into biliverdin-IX-alpha, it forms a powerful system involved in the cell stress response during neurodegenerative disorders. In this paper, an up-regulation of the biliverdin reductase-A protein levels was found in the hippocampus of the subjects with Alzheimer disease and arguably its earliest form, mild cognitive impairment. Moreover a significant reduction in the phosphorylation of serine, threonine and tyrosine residues of biliverdin reductase-A was found, and this was paralleled by a marked reduction in its reductase activity. Interestingly, the levels of both total and phosphorylated biliverdin reductase-A were unchanged as well as its enzymatic activity in the cerebella. These results demonstrated a dichotomy between biliverdin reductase-A protein levels and activity in the hippocampus of subjects affected by Alzheimer disease and mild cognitive impairment, and this effect likely is attributable to a reduction in the phosphorylation of serine, threonine and tyrosine residues of biliverdin reductase-A. Consequently, not just the increased levels of biliverdin reductase-A, but also its changed activity and phosphorylation state, should be taken into account when considering potential biomarkers for Alzheimer disease and mild cognitive impairment.► Increased biliverdin reductase (BVR) expression in AD and MCI hippocampus. ► Decreased phosphorylations (pSer/Thr and pTyr) on BVR in AD and MCI hippocampus. ► Decrease in BVR phosphorylation was associated with reduced activity. ► No change were observed in cerebellum, which lacks pathology in AD and MCI.

Keywords: Abbreviations; AD; Alzheimer disease; BVR; biliverdin reductase; HO; heme oxygenase; MCI; mild cognitive impairment; pSer/Thre; phospho-serine/threonine; pTyr; phospho-tyrosineAlzheimer disease; Biliverdin reductase; Cerebellum; Hippocampus; Mild cognitive impairment; Oxidative stress

Biophysical properties of mutant KCNQ1 S277L channels linked to hereditary long QT syndrome with phenotypic variability by Parwez Aidery; Jana Kisselbach; Patrick A. Schweizer; Rüdiger Becker; Hugo A. Katus; Dierk Thomas (pp. 488-494).

Hereditary long QT syndrome (LQTS) is associated with ventricular torsade de pointes tachyarrhythmias and sudden cardiac death. Mutations in a cardiac voltage-gated potassium channel, KCNQ1, induce the most frequent variant of LQTS. We identified a KCNQ1 missense mutation, KCNQ1 S277L, in a patient presenting with recurrent syncope triggered by emotional stress (QTc=528ms). This mutation is located in the conserved S5 transmembrane region of the KCNQ1 channel. Using in vitro electrophysiological testing in the Xenopus oocyte expression system, the S277L mutation was found to be non-functional and to suppress wild type currents in dominant-negative fashion in the presence and in the absence of the regulatory ß-subunit, KCNE1. In addition, expression of S277L and wild type KCNQ1 with KCNE1 resulted in a shift of the voltage-dependence of activation by −8.7mV compared to wild type I_Ks, indicating co-assembly of mutant and wild type subunits. The electrophysiological phenotype corresponds well with the severe clinical phenotype of the index patient. However, investigation of family members revealed three patients that exhibit asymptomatic QT interval prolongation (QTc=493–518ms). In conclusion, this study emphasizes the value of biophysical testing to provide mechanistic evidence for pathogenicity of ion channel mutations identified in LQTS patients. The inconsistent association of the KCNQ1 S277L mutation with the clinical presentation suggests that additional genetic, epigenetic, or environmental factors play a role in defining the individual clinical LQTS phenotype.► The KCNQ1 S277L mutation causes long QT syndrome 1. ► S277L subunits are non-functional. ► S277L suppresses wild type I_Ks in dominant-negative fashion. ► Modifying factors determine the individual clinical phenotype.

Keywords: Cardiac electrophysiology; I; _Ks; current; KCNQ1 (KVLQT1); Long QT syndrome; Mutation; Sudden cardiac death

Mechanism of lipid induced insulin resistance: Activated PKCε is a key regulator by Suman Dasgupta; Sushmita Bhattacharya; Sudipta Maitra; Durba Pal; Subeer S. Majumdar; Asis Datta; Samir Bhattacharya (pp. 495-506).

Fatty acids (FAs) are known to impair insulin signaling in target cells. Accumulating evidences suggest that one of the major sites of FAs adverse effect is insulin receptor (IR). However, the underlying mechanism is yet unclear. An important clue was indicated in leptin receptor deficient (d b/d b) diabetic mice where increased circulatory FAs was coincided with phosphorylated PKCε and reduced IR expression. We report here that central to this mechanism is the phosphorylation of PKCε by FAs. Kinase dead mutant of PKCε did not augment FA induced IRβ downregulation indicating phosphorylation of PKCε is crucial for FA induced IRβ reduction. Investigation with insulin target cells showed that kinase independent phosphorylation of PKCε by FA occurred through palmitoylation. Mutation at cysteine 276 and 474 residues in PKCε suppressed this process indicating participation of these two residues in palmitoylation. Phosphorylation of PKCε endowed it the ability to migrate to the nuclear region of insulin target cells. It was intriguing to search about how translocation of phosphorylated PKCε occurred without having canonical nuclear localization signal (NLS). We found that F-actin recognized phospho-form of PKCε and chaperoned it to the nuclear region where it interact with HMGA1 and Sp1, the transcription regulator of IR and HMGA1 gene respectively and impaired HMGA1 function. This resulted in the attenuation of HMGA1 driven IR transcription that compromised insulin signaling and sensitivity.► This work describes the molecular mechanism of lipid induced insulin resistance. ► Fatty acid (FA) downregulates insulin receptor expression in insulin target cells. ► FA phosphorylates PKCε (pPKCε) that adversely affects insulin receptor (IR). ► pPKCε impairs HMGA1, the transcription regulator of IR gene. ► Reduced IR expression due to FA abrogates insulin sensitivity.

Keywords: Fatty acid; HMGA1; Insulin resistance; IRβ; PKCε

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons by Marcus J. Calkins; P. Hemachandra Reddy (pp. 507-513).

Loss of synapses and synaptic damage are the best correlates of cognitive decline identified in patients with Alzheimer's disease (AD), and mitochondrial oxidative damage and synaptic pathology have been identified as early events in the progression of AD. The progressive accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria are hypothesized to cause synaptic degeneration and cognitive decline in patients with AD. However, the precise mechanistic link between Aβ and mitochondria is not well understood. The purpose of this study was to better understand the effects of Aβ on mitochondrial axonal transport and synaptic alterations in AD. Using mouse hippocampal neurons and Aβ_25–35 peptide, we studied axonal transport of mitochondria, including mitochondrial motility, mitochondrial length and size, mitochondrial index per neurite, and synaptic alterations of the hippocampal neurons. In the PBS-treated neurons, 36.4±4.7% of the observed mitochondria were motile, with 21.0±1.3% moving anterograde and 15.4±3.4% moving retrograde and the average speed of movement was 12.1±1.8μm/min. In contrast, in the Aβ-treated neurons, the number of motile mitochondria were significantly less, at 20.4±2.6% (P<0.032), as were those moving anterograde (10.1±2.6%, P<0.016) relative to PBS-treated neurons, suggesting that the Aβ_25–35 peptide impairs axonal transport of mitochondria in AD neurons. In the Aβ-treated neurons, the average speed of motile mitochondria was also less, at 10.9±1.9μm/min, and mitochondrial length was significantly decreased. Further, synaptic immunoreactivity was also significantly less in the Aβ-treated neurons relative to the PBS-treated neurons, indicating that Aβ affects synaptic viability. These findings suggest that, in neurons affected by AD, Aβ is toxic, impairs mitochondrial movements, reduces mitochondrial length, and causes synaptic degeneration.► Mitochondrial transport and synaptic viability were studied in Aβ-treated hippocampal neurons. ► Mitochondrial mass and axonal transport were reduced in Aβ-treated neurons. ► Increased mitochondrial fission and reduced synaptic markers were found. ► We conclude that Aβ impairs axonal transport of mitochondria and may cause synaptic degeneration.

Keywords: Abbreviations; AβPP; Aβ precursor protein; AD; Alzheimer's disease; ATP; adenosine triphosphate; Drp1; dynamin related protein 1; Fis1; mitochondrial fission 1Mitochondria; Axonal transport; Alzheimer's disease; Mitochondrial dynamics; Mitochondrial fission; Amyloid beta

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons by Marcus J. Calkins; P. Hemachandra Reddy (pp. 507-513).

O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy by Wenyi Mi; Yuchao Gu; Cuifang Han; Haiyan Liu; Qiong Fan; Xinling Zhang; Qi Cong; Wengong Yu (pp. 514-519).

O-GlcNAc is a monosaccharide attached to serine or threonine hydroxyl moieties on numerous nuclear and cytoplasmic proteins; O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Although recent studies have shown that O-GlcNAcylation plays essential roles in breast cancer progression, it is also necessary to know whether O-GlcNAcylation is involved in other types of human cancer. In this study, O-GlcNAcylation levels and the expressions of OGT and OGA in human lung and colon cancer tissues were examined by immunohistochemistry analysis. We found that O-GlcNAcylation as well as OGT expression was significantly elevated in the cancer tissues compared with that in the corresponding adjacent tissues. Additionally, the roles of O-GlcNAcylation in the malignancy of lung and colon cancer were investigated in vitro. The results showed that O-GlcNAcylation markedly enhanced the anchorage-independent growth of lung and colon cancer cells; O-GlcNAcylation could also enhance lung and colon cancer invasion in a context-dependent manner. All together, this study suggests that O-GlcNAcylation might play important roles in lung and colon cancer formation and progression, and may be a valuable target for diagnosis and therapy of cancer.►The levels of O-GlcNAc and OGT were elevated in the lung and colon cancer tissues. ►OGT increase may be a main cause of O-GlcNAc elevation in tumor tissues. ►O-GlcNAc plays important roles in lung and colon cancer malignancy.

Keywords: O-GlcNAc; Lung cancer; Colon cancer; Immunohistochemistry

O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy by Wenyi Mi; Yuchao Gu; Cuifang Han; Haiyan Liu; Qiong Fan; Xinling Zhang; Qi Cong; Wengong Yu (pp. 514-519).

Keywords: O-GlcNAc; Lung cancer; Colon cancer; Immunohistochemistry

Zebrafish as a model to understand autophagy and its role in neurological disease by Angeleen Fleming; David C. Rubinsztein (pp. 520-526).

In the past decade, the zebrafish ( Danio rerio) has become a popular model system for the study of vertebrate development, since the embryos and larvae of this species are small, transparent and undergo rapid development ex utero, allowing in vivo analysis of embryogenesis and organogenesis. These characteristics can also be exploited by researchers interested in signaling pathways and disease processes and, accordingly, there is a growing literature on the use of zebrafish to model human disease. This model holds great potential for exploring how autophagy, an evolutionarily conserved mechanism for protein degradation, influences the pathogeneses of a range of different human diseases and for the evaluation of this pathway as a potential therapeutic strategy. Here we summarize what is known about the regulation of autophagy in eukaryotic cells and its role in neurodegenerative disease and highlight how research using zebrafish has helped further our understanding of these processes.► Zebrafish has become a popular model system for the study of vertebrate development. ► There is a growing literature on the use of zebrafish to model human disease. ► This model has potential for exploring how autophagy affects disease pathogenesis. ► This review considers how zebrafish may help further understanding of these processes.

Keywords: Zebrafish; Autophagy; Neurodegeneration

Zebrafish as a model to understand autophagy and its role in neurological disease by Angeleen Fleming; David C. Rubinsztein (pp. 520-526).

Keywords: Zebrafish; Autophagy; Neurodegeneration

Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes by Libin Zhou; Xiao Wang; Ying Yang; Ling Wu; Fengying Li; Rong Zhang; Guoyue Yuan; Ning Wang; Mingdao Chen; Guang Ning (pp. 527-535).

Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of³[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.

Keywords: Abbreviations; FFA; free fatty acid; HSL; hormone-sensitive lipase; PDE; phosphodiesterase; TNF-α; tumor necrosis factor α; AMPK; AMP-activated protein kinase; ACC; acetyl-CoA carboxylase; PKA; protein kinase A; ERK; extracellular signal-regulated kinase; MAPK; mitogen-activated protein kinase; PI3K; phosphoinositide 3-kinaseBerberine; cAMP signaling; Phosphodiesterase; AMPK; Lipolysis; Adipocytes

Pathogenic effects of a novel mutation (c.664_681del) in KCNQ4 channels associated with auditory pathology by Jeong-In Baek; Hong-Joon Park; Kyungjoon Park; Su-Jin Choi; Kyu-Yup Lee; Jee Hyun Yi; Thomas B. Friedman; Dennis Drayna; Ki Soon Shin; Un-Kyung Kim (pp. 536-543).

Hearing loss is a common communication disorder caused by various environmental and genetic factors. Hereditary hearing loss is very heterogeneous, and most of such cases involve sensorineural defects in the auditory pathway. There are currently 57 known autosomal dominant non-syndromic hearing loss (DFNA) loci, and the causative genes have been identified at 22 of these loci. In the present study, we performed a genome-wide linkage analysis in a Korean family segregating autosomal dominant hearing loss. We observed linkage on chromosome 1p34, and at this locus, we detected a novel mutation consisting of an 18 nucleotide deletion in exon 4 of the KCNQ4 gene, which encodes a voltage-gated potassium channel. We carried out a functional in vitro study to analyze the effects of this mutation (c.664_681del) along with two previously described KCNQ4 mutations, p.W276S and p.G285C. Although the c.664_681del mutation is located in the intercellular loop and the two previously described mutations, p.W276S and p.G285C, are located in the pore region, all mutants inhibit normal channel function by a dominant negative effect. Our analysis indicates that the intercellular loop is as significant as the pore region as a potential site of pathogenic effects on KCNQ4 channel function.►Detection a novel mutation consisting of an 18 nucleotide deletion in exon 4 of the KCNQ4 gene in an autosomal dominant non-syndromic hearing loss family. ►A functional in vitro study to analyze the effects of this mutation (c.664_681del) along with two previously described KCNQ4 mutations, p.W276S and p.G285C. ►All mutants inhibit normal channel function by a dominant negative effect and it indicates that the intercellular loop is as significant as the pore region as a potential site of pathogenic effects on KCNQ4 channel function.

Keywords: Hearing loss; KCNQ4; K; ⁺; channel; Mutation; Dominant negative effect

Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration by Barry B. Muhoberac; Martin A. Baraibar; Ruben Vidal (pp. 544-548).

Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.► A mutant ferritin light chain (FTL) with altered C-terminal sequence causes the neurodegenerative disease hereditary ferritinopathy (HF). ► Mutant FTLs undergo facile assembly with wild-type ferritin heavy chains (or wild-type FTLs) forming dysfunctional ferritin shells that exhibit iron loading-induced ferritin aggregation and decreased iron incorporation. ► Formation of aggregates may help explain inclusion bodies (IBs) found in the brain of HF patents and can be explained by our model of iron bridging between unraveled and extended mutant FTL C-termini on different shells. ► Decreased iron incorporation into soluble ferritin follows from unraveled mutant FTL C-termini causing leaky and disordered 4-fold ferritin pores. ► Our in vitro results are consistent with cellular abnormalities such as IB formation, iron mishandling, and free radical damage in HF, which may be explained in terms of a loss of normal function and gain of toxic function of ferritin.

Keywords: Abbreviations; FTL; ferritin light chain; FTH1; (wild-type) ferritin heavy chain; Wt-FTL; wild-type ferritin light chain; Mt-FTL; mutant ferritin light chain; HF; hereditary ferritinopathy; IBs; inclusion bodies; GdnHCl; guanidine hydrochloride; ROS; reactive oxygen speciesFerritin; Iron; Pore; Neurodegeneration; Hereditary ferritinopathy

Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion by Mien V. Hoang; Lois E.H. Smith; Donald R. Senger (pp. 549-557).

In ischemic retinopathies, underlying hypoxia drives abnormal neovascularization that damages retina and causes blindness. The abnormal neovasculature is tortuous and leaky and fails to alleviate hypoxia, resulting in more pathological neovascularization and retinal damage. With an established model of ischemic retinopathy we found that calpain inhibitors, when administered in moderation, reduced architectural abnormalities, reduced vascular leakage, and most importantly reduced retinal hypoxia. Mechanistically, these calpain inhibitors improved stability and organization of the actin cytoskeleton in retinal endothelial cells undergoing capillary morphogenesis in vitro, and they similarly improved organization of actin cables within new blood vessels in vivo. Hypoxia induced calpain activity in retinal endothelial cells and severely disrupted the actin cytoskeleton, whereas calpain inhibitors preserved actin cables under hypoxic conditions. Collectively, these findings support the hypothesis that hyper-activation of calpains by hypoxia contributes to disruption of the retinal endothelial cell cytoskeleton, resulting in formation of neovessels that are defective both architecturally and functionally. Modest suppression of calpain activity with calpain inhibitors restores cytoskeletal architecture and promotes formation of a functional neovasculature, thereby reducing underlying hypoxia. In sharp contrast to “anti-angiogenesis” strategies that cannot restore normoxia and may aggravate hypoxia, the therapeutic strategy described here does not inhibit neovascularization. Instead, by improving the function of neovascularization to reduce underlying hypoxia, moderate calpain inhibition offers a method for alleviating retinal ischemia, thereby suggesting a new treatment paradigm based on improvement rather than inhibition of new blood vessel growth.►Hypoxia activates calpain in retinal endothelial cells. ►Calpain inhibitors, at suitable doses, improve the architecture of hypoxia-induced retinal neovessels. ►With optimal doses of calpain inhibitors, retinal neovessels exhibit reduced leakiness. ►Improved retinal neovascularization results in reduced retinal hypoxia. ►Optimal administration of calpain inhibitors likely improves neovessel architecture by stabilizing actin.

Keywords: Calpain; Retinopathy; Neovascularization; Hypoxia; Endothelial cell; Cytoskeleton

Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion by Mien V. Hoang; Lois E.H. Smith; Donald R. Senger (pp. 549-557).

Keywords: Calpain; Retinopathy; Neovascularization; Hypoxia; Endothelial cell; Cytoskeleton

Fenofibrate exhibits a high potential to suppress the formation of squamous cell carcinoma in an oral-specific 4-nitroquinoline 1-oxide/arecoline mouse model by Nai Wen Chang; Ming-Hsui Tsai; Chingju Lin; Hui Ting Hsu; Pei-Yi Chu; Chung-Min Yeh; Chang-Fang Chiu; Kun-Tu Yeh (pp. 558-564).

The excessive use of areca nut and/or tobacco may induce the production of free radicals and reactive oxygen species, which affect the lipid contents of the cell membrane and are possibly involved in tumorigenic processes in the oral cavity. The aim of this study was to investigate the therapeutic efficacy of fenofibrate (0.1% or 0.3%, w/w), a ligand of the peroxisome proliferator-activated receptor alpha (PPARα), in a 4-nitroquinoline 1-oxide (4-NQO)/arecoline-induced oral cancer mouse model. The carcinogen, 4-NQO/arecoline, was administrated to C57BL/6JNarl mice for 8weeks followed by fenofibrate treatment for 12 or 20weeks. After 28weeks, changes in serum lipids, the multiplicity of tumor lesions, and tumor sizes were determined together with changes in the immunohistochemical expressions of PPARα, acetyl-coenzyme A carboxylase (ACC), the epidermal growth factor receptor (EGFR), and cyclooxygenase-2 (COX2). The results showed that when compared to the 4-NQO/arecoline only group, 0.3% fenofibrate treatment increased serum total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels. 0.3% fenofibrate treatment suppressed the incidence rate of tongue lesions, reduced the multiplicity of squamous cell carcinoma (SCC), decreased the tumor size, and increased the immunoreactivity of EGFR and COX2 in oral dysplasia but decreased EGFR and COX2 expressions in SCC. These findings indicated that fenofibrate reduced the tumor incidence rate and suppressed the tumor progression into SCC and that these molecular events might be linked to the EGFR and COX2 regulatory pathways. We suggest that fenofibrate provides a new strategy for preventing oral tumor progression.►Fenofibrate has a high potential to inhibit the formation of squamous cell carcinoma. ►The effects of fenofibrate might be linked to the EGFR and COX2 regulatory pathways. ►Fenofibrate provides a new strategy for preventing tongue tumor progression.

Keywords: Abbreviations; ACC; acetyl-coenzyme A carboxylase; BQ; betel quid; COX2; cyclooxygenase-2; EGFR; epidermal growth factor receptor; FAS; fatty acid synthase; HDL-C; high-density lipoprotein cholesterol; LDL-C; low-density lipoprotein cholesterol; 4-NQO; 4-nitroquinoline 1-oxide; PPARα; peroxisome proliferator-activated receptor alpha; SCC; squamous cell carcinoma; TC; total cholesterol; VLDL; very low-density lipoproteinFenofibrate; 4-Nitroquinoline 1-oxide; Arecoline; Oral squamous cell carcinoma

Tumor necrosis factor-alpha regulates the Hypocretin system via mRNA degradation and ubiquitination by Shuqin Zhan; Guo-Qiang Cai; Anni Zheng; Yuping Wang; Jianping Jia; Haotian Fang; Youfeng Yang; Meng Hu; Qiang Ding (pp. 565-571).

Recent studies recognize that Hypocretin system (also known as Orexin) plays a critical role in sleep/wake disorders and feeding behaviors. However, little is known about the regulation of the Hypocretin system. It is also known that tumor necrosis factor alpha (TNF-α) is involved in the regulation of sleep/wake cycle. Here, we test our hypothesis that the Hypocretin system is regulated by TNF-α. Prepro-Hypocretin and Hypocretin receptor 2 (HcrtR2) can be detected at a very low level in rat B35 neuroblastoma cells. In response to TNF-α, Prepro-Hypocretin mRNA and protein levels are down-regulated, and also HcrtR2 protein level is down-regulated in B35 cells. To investigate the mechanism, exogenous rat Prepro-Hypocretin and rat HcrtR2 were overexpressed in B35 cells. In response to TNF-α, protein and mRNA of Prepro-Hypocretin are significantly decreased (by 93% and 94%, respectively), and the half-life of Prepro-Hypocretin mRNA is decreased in a time- and dose-dependent manner. The level of HcrtR2 mRNA level is not affected by TNF-α treatment; however, HcrtR2 protein level is significantly decreased (by 86%) through ubiquitination in B35 cells treated with TNF-α. Downregulation of cellular inhibitor of apoptosis protein-1 and -2 (cIAP-1 and -2) abrogates the HcrtR2 ubiquitination induced by TNF-α. The control green fluorescent protein (GFP) expression is not affected by TNF-α treatment. These studies demonstrate that TNF-α can impair the function of the Hypocretin system by reducing the levels of both Prepro-Hypocretin and HcrtR2.▶ The pro-inflammation cytokine, TNF-alpha, impairs the Hypocretin/Orexin system. ▶ TNF-alpha decreases the expression of Prepro-Hypocretin and Hypocretin receptor 2. ▶ TNF-alpha increases mRNA degradation and ubiquitination of the Hypocretin system. ▶ Impaired Hypocretin system contributes to the development of sleep disorders. ▶ TNF-alpha may regulate the sleep-wake cycle through the Hypocretin system.

Keywords: Abbreviations; TNF-α; tumor necrosis factor alpha; HcrtR2; Hypocretin receptor 2; cIAP; cellular inhibitor of apoptosis protein; FAK; focal adhesion kinase; G3PDH; glyceraldehyde 3-phosphate dehydrogenase; RT-PCR; reverse transcriptase polymerase chain reaction; MSCV vector; murine stem cell viral vector; GFP; green-fluorescent-protein; mRNA; messenger RNAHypocretin; Orexin; Tumor necrosis factor; Sleep disorder; Hypocretin receptor; Orexin receptor

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BBA - Molecular Basis of Disease (v.1812, #4)