BBA - Molecular Basis of Disease (v.1822, #8)

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome.▶ NADPH is an essential reducing equivalent for fat and cholesterol biosynthesis. ▶ IDPc has been proposed as a key enzyme for supplying cytosolic NADPH. ▶ Knockdown of IDPc by RNA interference inhibited adipogenesis and lipid biosynthesis.
Keywords: NADPH; Obesity; RNAi; Adipogenesis; Lipogenesis;

Impaired protein quality control system underlies mitochondrial dysfunction in skeletal muscle of streptozotocin-induced diabetic rats by Ana Isabel Padrão; Tiago Carvalho; Rui Vitorino; Renato M.P. Alves; Armando Caseiro; José Alberto Duarte; Rita Ferreira; Francisco Amado (1189-1197).
Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality.Display Omitted► After 4 months of STZ administration gastrocnemius IMF mitochondria functionality decreased. ► Increased mitochondria carbonylation levels were paralleled by decreased OXPHOS activity. ► Decreased mitochondria proteolysis was related with increased protein oxidation. ► AAA proteases activity and expression decreased in diabetic animals.
Keywords: Intermyofibrillar mitochondria; Mitochondrial proteolysis; Protein quality control; Gastrocnemius; Type 1 diabetes mellitus;

Herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-phosphofructo-1-kinase (PFK-1) by Juliana L. Abrantes; Cristiane M. Alves; Jessica Costa; Fabio C.L. Almeida; Mauro Sola-Penna; Carlos Frederico L. Fontes; Thiago Moreno L. Souza (1198-1206).
Viruses such as HIV, HCV, Mayaro and HCMV affect cellular metabolic pathways, including glycolysis. Although some studies have suggested that the inhibition of glycolysis affects HSV-1 replication and that HSV-1-infected eyes have increased lactate production, the mechanisms by which HSV-1 induces glycolysis have never been investigated in detail. In this study, we observed an increase in glucose uptake, lactate efflux and ATP content in HSV-1-infected cells. HSV-1 triggered a MOI-dependent increase in the activity of phosphofructokinase-1 (PFK-1), a key rate-limiting enzyme of the glycolytic pathway. After HSV-1 infection, we observed increased PFK-1 expression, which increased PFK-1 total activity, and the phosphorylation of this enzyme at serine residues. HSV-1-induced glycolysis was associated with increased ATP content, and these events were critical for viral replication. In summary, our results suggest that HSV-1 triggers glycolysis through a different mechanism than other herpesviruses, such as HCMV. Thus, this study contributes to a better understanding of HSV-1 pathogenesis and provides insights into novel targets for antiviral therapy.►HSV-1 activates glycolysis by PFK-1 activation. ►In HSV-1-infected cells PFK-1 synthesis is up-regulated and phosphorylated at serine residues. ►PFK-1 knockdown impairs HSV-1 replication. ►HSV-1-mediated glycolysis activation increases ATP content.
Keywords: HSV-1; PFK-1; Glycolysis;

Inhibitory effect of curcumin on the Al(III)-induced Aβ42 aggregation and neurotoxicity in vitro by Teng Jiang; Xiu-Ling Zhi; Yue-Hong Zhang; Luan-Feng Pan; Ping Zhou (1207-1215).
The pathogenesis of Alzheimer's disease (AD) involves a key event which changes the morphology of amyloid-β 42 (Aβ42) peptide from its soluble monomeric form into the fibrillated aggregates in the brain. Aluminum ion, Al(III), is known to act as a pathological chaperone of the Aβ42 in this process; curcumin, a natural phenolic compound, is considered capable of binding Al(III) and Aβ42; nevertheless, little is known about the combined action of curcumin and Al(III) on the Aβ42 fibrillation and neurotoxicity. Here, combinations of circular dichroism spectroscopy, thioflavin T fluorescence, atomic force microscopy, Bradford and MTT assays, it is demonstrated that although Al(III) can promote the Aβ42 fibrillation dose-dependently, leading to the high neurotoxicity to PC12 cells, curcumin can inhibit the events. Besides, we found that curcumin is able not only to inhibit the formation of Al(III)-induced Aβ42 fibrillation, but also to form the Al(III)–curcumin complexes which in turn can remold the preformed, mature, ordered Aβ42 fibrils into the low toxic amorphous aggregates. These findings suggest that curcumin could block the binding of Al(III) with Aβ42 and form the Al(III)–curcumin complexes, so as to inhibit the Al(III)-induced Aβ42 fibrillation and neurotoxicity. The Al(III)–curcumin complexes are worth potentially developing as a therapy agent against the neurodegenerative disorders in the future.Curcumin is able not only to inhibit the formation of Al(III)-induced Aβ42 fibrillation, but also to form the Al(III)–curcumin complexes which in turn can remold the preformed, mature, ordered Aβ42 fibrils into the low toxic amorphous aggregates.Display Omitted► Al(III) promotes Aβ42 fibrillation, leading to the high neurotoxicity to PC12 cells. ► Curcumin is able to inhibit the formation of Al(III)-induced Aβ42 fibrillation. ► Al(III)–curcumin complexes can remold the Aβ42 fibrils into amorphous aggregates.
Keywords: Alzheimer's disease; Protein conformation; Aggregate morphology; Fibrillation inhibitor;

Oxidative phosphorylation differences between mitochondrial DNA haplogroups modify the risk of Leber's hereditary optic neuropathy by Aurora Gómez-Durán; David Pacheu-Grau; Íñigo Martínez-Romero; Ester López-Gallardo; Manuel J. López-Pérez; Julio Montoya; Eduardo Ruiz-Pesini (1216-1222).
Leber's hereditary optic neuropathy is a maternally inherited optic atrophy caused by mitochondrial DNA point mutations. Previous epidemiological studies have shown that individuals from mitochondrial genetic backgrounds (haplogroups) J/Uk and H have a higher and a lower risk, respectively, of suffering this disorder. To analyze the bases of these associations at cellular and molecular levels, functional studies with cybrids provide high quality evidence. Cybrids from haplogroup J contain less mitochondrial deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and synthesize a smaller amount of mitochondrial DNA-encoded polypeptides than those from haplogroup H. Haplogroup J cybrids also display lower oxygen consumption, mitochondrial inner membrane potential and total adenosine‐5′‐triphosphate (ATP) levels. Moreover, mitochondrial DNA levels correlate with many parameters of the oxidative phosphorylation system. These results suggest that the mitochondrial DNA amount determines oxidative phosphorylation capacity and, along with other recently published observations, support the possibility that mitochondrial DNA levels may be responsible for the bias of the disorder toward males, for the incomplete penetrance of mutations causing Leber's hereditary optic neuropathy and for the association of the disease with particular mitochondrial DNA haplogroups.► MtDNA haplogroup J and H mutation carriers have a different risk of suffering LHON. ► Cybrids allow the analysis of the cellular and molecular bases for the risk of LHON. ► MtDNA haplogroup J and H cybrids show phenotypic differences in OXPHOS function. ► MtDNA levels determine OXPHOS capacity. ► MtDNA levels may be responsible for the association of LHON with mtDNA haplogroups.
Keywords: Leber's hereditary optic neuropathy; Oxidative phosphorylation; Mitochondrial DNA; Haplogroup; Cybrid;

Ezetimibe: A biomarker for efficacy of liver directed UGT1A1 gene therapy for inherited hyperbilirubinemia by Paula S. Montenegro-Miranda; Nina Sneitz; D. Rudi de Waart; Lysbeth ten Bloemendaal; Suzanne Duijst; Robert J. de Knegt; Ulrich Beuers; Moshe Finel; Piter J. Bosma (1223-1229).
As recently demonstrated in patients with factor IX deficiency, adeno‐associated virus (AAV)-mediated liver-directed therapy is a viable option for inherited metabolic liver disorders. Our aim is to treat Crigler-Najjar syndrome type I (CN I), an inherited severe unconjugated hyperbilirubinemia, as a rare recessive inherited disorder. Because the number of patients eligible for this approach is small, the efficacy can only be demonstrated by a beneficial effect on the pathophysiology in individual patients. Serum bilirubin levels in potential candidates have been monitored since birth, providing an indication of their pathophysiology. Adjuvant phototherapy to prevent brain damage reduces serum unconjugated bilirubin (UCB) levels in CN I patients to the level seen in the milder form of the disease, CN type II. This therapy increases the excretion of UCB, thereby complicating the use of UCB and conjugated bilirubin levels in serum as biomarkers for the gene therapy we try to develop. Therefore, a suitable biomarker that is not affected by phototherapy is currently needed. To this end, we have investigated whether estradiol, ethinylestradiol or ezetimibe could be used as markers for uridine 5'-di-phospho-glucuronosyltransferase isoform 1A1 (UGT1A1) activity restored by AAV gene therapy in Gunn rats, a relevant animal model for CN I. Of these compounds, ezetimibe appeared most suitable because its glucuronidation rate in untreated control Gunn rats is low. Subsequently, ezetimibe glucuronidation was studied in both untreated and AAV-treated Gunn rats and the results suggest that it may serve as a useful serum marker for restored hepatic UGT1A1 activity.► Ezetimibe glucuronidation is a biomarker for UGT1A1 activity in vivo. ► Ezetimibe glucuronidation correlates with reduction of serum bilirubin in Gunn rats. ► Ezetimibe glucuronidation in the Gunn rat is affected by gender. ► Bilirubin glucuronides in bile confirm efficacy of gene therapy for inherited jaundice.
Keywords: bilirubin; self‐complementary adeno‐associated viral vector; liver; biomarker; gene therapy;

Development of diet-induced insulin resistance in adult Drosophila melanogaster by Siti Nur Sarah Morris; Claire Coogan; Khalil Chamseddin; Sun Ok Fernandez-Kim; Santharam Kolli; Jeffrey N. Keller; Johannes H. Bauer (1230-1237).
The fruit fly Drosophila melanogaster is increasingly utilized as an alternative to costly rodent models to study human diseases. Fly models exist for a wide variety of human conditions, such as Alzheimer's and Parkinson’s Disease, or cardiac function. Advantages of the fly system are its rapid generation time and its low cost. However, the greatest strength of the fly system are the powerful genetic tools that allow for rapid dissection of molecular disease mechanisms. Here, we describe the diet-dependent development of metabolic phenotypes in adult fruit flies. Depending on the specific type of nutrient, as well as its relative quantity in the diet, flies show weight gain and changes in the levels of storage macromolecules. Furthermore, the activity of insulin-signaling in the major metabolic organ of the fly, the fat body, decreases upon overfeeding. This decrease in insulin-signaling activity in overfed flies is moreover observed when flies are challenged with an acute food stimulus, suggesting that overfeeding leads to insulin resistance. Similar changes were observed in aging flies, with the development of the insulin resistance-like phenotype beginning at early middle ages. Taken together, these data demonstrate that imbalanced diet disrupts metabolic homeostasis in adult D. melanogaster and promotes insulin-resistant phenotypes. Therefore, the fly system may be a useful alternative tool in the investigation of molecular mechanisms of insulin resistance and the development of pharmacologic treatment options. ► D. melanogaster as a model for diet-induced insulin resistance ► Overfed adult fruit flies develop diet-dependent metabolic abnormalities. ► Overfed flies furthermore develop insulin resistance. ► The fly model for disrupted metabolic homeostasis will be useful for investigating novel treatment options.
Keywords: Diabetes; Drosophila melanogaster; Insulin signaling; Insulin resistance; Diet;

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels by Edgar Garza-López; Alejandro Sandoval; Ricardo González-Ramírez; María A. Gandini; Arn Van den Maagdenberg; Michel De Waard; Ricardo Felix (1238-1246).
Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of CaV2.1 voltage-gated Ca2 + channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human CaV2.1α1 subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK‐293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant CaV2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca2 + channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.► Familial hemiplegic migraine type 1 (FHM-1) is a subtype of migraine with aura. ► FHM-1 is caused by missense mutations in the CACNA1A gene. ► CACNA1A encodes the CaV2.1 voltage-gated Ca2 + channels. ► G protein regulation of FHM-1 mutant channels W1684R and V1696I was investigated. ► G protein-Ca2 + channel affinity was altered by the FHM-1 mutations.
Keywords: Migraine; Familial hemiplegic migraine; FHM-1; Calcium channel; G-protein regulation;

Role of hippocalcin in mediating Aβ toxicity by Yun-An Lim; Maria Giese; Claire Shepherd; Glenda Halliday; Masaaki Kobayashi; Ken Takamatsu; Matthias Staufenbiel; Anne Eckert; Jürgen Götz (1247-1257).
Alzheimer's disease (AD) is the most common cause of dementia, and amyloid-β (Aβ) plaques and tau-containing tangles are its histopathological hallmark lesions. These do not occur at random; rather, the neurodegenerative process is stereotyped in that it is initiated in the entorhinal cortex and hippocampal formation. Interestingly, it is the latter brain area where the calcium-sensing enzyme hippocalcin is highly expressed. Because calcium deregulation is a well-established pathomechanism in AD, we aimed to address the putative role of hippocalcin in human AD brain and transgenic mouse models. We found that hippocalcin levels are increased in human AD brain and in Aβ plaque-forming APP23 transgenic mice compared to controls. To determine the role of hippocalcin in Aβ toxicity, we treated primary cultures derived from hippocalcin knockout (HC KO) mice with Aβ and found them to be more susceptible to Aβ toxicity than controls. Likewise, treatment with either thapsigargin or ionomycin, both known to deregulate intracellular calcium levels, caused an increased toxicity in hippocampal neurons from HC KO mice compared to wild-type. We found further that mitochondrial complex I activity increased from 3 to 6 months in hippocampal mitochondria from wild-type and HC KO mice, but that the latter exhibited a significantly stronger aging phenotype than wild-type. Aβ treatment induced significant toxicity on hippocampal mitochondria from HC KO mice already at 3 months of age, while wild-type mitochondria were spared. Our data suggest that hippocalcin has a neuroprotective role in AD, presenting it as a putative biomarker.► The calcium sensor hippocalcin is deregulated in Alzheimer's disease hippocampus. ► In APP mutant mice, hippocalcin levels are increased. ► In these mice, hippocampal neurons that are high in Abeta are low in hippocalcin. ► Hippocampal neurons from hippocalcin KO mice are sensitive to calcium deregulators. ► Cortical neurons from hippocalcin KO mice show sensitivity similar to wild-type. ► Hippocalcin knock-out neurons show an age-dependent mitochondrial deregulation.
Keywords: Alzheimer; Amyloid; Calcium; Pathogenesis; Toxicity; Transgenic mice;

Targeted knockdown of Cerkl, a retinal dystrophy gene, causes mild affectation of the retinal ganglion cell layer by Alejandro Garanto; Javier Vicente-Tejedor; Marina Riera; Pedro de la Villa; Roser Gonzàlez-Duarte; Román Blanco; Gemma Marfany (1258-1269).
In order to approach the function of the retinal dystrophy CERKL gene we generated a novel knockout mouse model by cre-mediated targeted deletion of the Cerkl first exon and proximal promoter. The excised genomic region (2.3 kb) encompassed the first Cerkl exon, upstream sequences including the proximal promoter and the initial segment of the first intron. The Cerkl −/− mice were viable and fertile. The targeted Cerkl deletion resulted in a knockdown more than a knockout model, given that alternative promoters (unreported at that time) directed basal expression of Cerkl (35%). In situ hybridizations and immunohistochemistry showed that this remnant expression was moderate in the photoreceptors and weak in the ganglion and inner cell layers. Morphological characterization of the Cerkl −/− retinas did not show any gross structural changes, even at 12 months of age. However, some clear and consistent signals of gliosis and retinal stress were detected by the statistically significant increase of i) the glial fibrillary antigen protein (GFAP) expression, and ii) apoptosis, as detected by TUNEL. Remarkably, consistent non-progressive perturbation (from birth up to 12 months of age) of ganglion cells was supported by the decrease of the Brn3a marker expression as well as the reduced oscillatory potentials in the electroretinographic recordings. In conclusion, the Cerkl −/− knockdown shows a mild retinal phenotype, with increased levels of cellular stress and apoptosis indicators, and clear signs of functional alteration at the ganglion cell layer, but no detectable morphological changes.► Cerkl-/- knockout mice were generated by cre-mediated excision of the first exon and proximal promoter region. ► Previously unreported alternative promoters directed basal expression of Cerkl (35%). ► No gross morphological alteration was evident in the Cerkl-/- retinas. ► Retinal stress and functional alteration was supported by an increase of apoptosis and GFAP, and a decrease of Brn3a. ► ERG recordings showed consistent perturbation of the oscillatory potentials, supporting ganglion cell alteration.
Keywords: Knockout model; Retinal dystrophy; Retinitis pigmentosa; Ganglion cell;

Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells by Martina Malnar; Marko Kosicek; Ana Lisica; Melanija Posavec; Ana Krolo; Jasenka Njavro; Damir Omerbasic; Sabina Tahirovic; Silva Hecimovic (1270-1283).
Cholesterol accumulation in Niemann–Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-β peptide (Aβ), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by β-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682–691.). We hypothesized that increased formation of APP-CTFs and Aβ in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aβ levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased β-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.►We examine the mechanism of increased β-secretase processing of APP in NPC1 −/− cells. ►APP internalization is prerequisite for increased Aβ/APP-CTF levels in NPC1 −/− cells. ►Upon NPC1-loss APP and BACE1 (β-secretase) sequester within endocytic vesicles. ►Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1 −/− cells. ►Cholesterol modulates Alzheimer's disease by altering trafficking of APP and BACE1.
Keywords: Alzheimer's disease; APP; BACE1; Cholesterol; Endocytosis; NPC1;

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila by Ema Alves; Bárbara J. Henriques; João V. Rodrigues; Pedro Prudêncio; Hugo Rocha; Laura Vilarinho; Rui G. Martinho; Cláudio M. Gomes (1284-1292).
Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of β-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a β-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD.Display Omitted► Fly mutants defective for embryonic cuticle allelic to ETF:QO were identified. ► Three missense mutations identified in ETF:QO (G65E, A68V and S104F). ► Raised acylcarnitine levels in fly embryos as in severe MADD. ► Mutations map at FAD motif within Rossmann fold inactivating enzyme. ► Severe MADD-like phenotype in the fruit fly sets proof of concept of suitability as disease model.
Keywords: Flavoprotein; Drosophila; Multiple acyl-CoA dehydrogenase deficiency (MADD); Inherited metabolic defect; Rossmann fold; Acylcarnitines;

A new mouse model for studying EGFR-dependent gastric polyps by Maik Dahlhoff; Markus Gerhard; Roland Rad; Sara Lindén; Eckhard Wolf; Marlon R. Schneider (1293-1299).
Hyperactivation of the epidermal growth factor receptor (EGFR) in gastric cells due to excess of its ligand transforming growth factor-α (TGFA) is associated with hyperplastic lesions in Ménétrier's disease patients and in transgenic mice. Other EGFR ligands, however, have never been associated with stomach diseases. Here, we report that overexpression of the EGFR ligand betacellulin (BTC) results in a severe, age-dependent hyperplasia of foveolar epithelium. The stomach weight of affected mice reached up to 3 g representing more than 10% of total body weight. The preexisting corpus mucosa was severely depleted, and both parietal and chief cells were replaced by proliferating foveolar epithelium. The lesions were more severe in male as compared to female transgenic mice, and partially regressed in the former after castration-mediated androgen removal. The gastric hyperplasia fully disappeared when BTC-tg mice were crossed into the Egfr Wa5 background expressing a dominant-negative EGFR, indicating that the phenotype is EGFR-dependent. This is, to our knowledge, the first report of hyperplastic gastric lesions due to the overexpression of an EGFR ligand other than TGFA. BTC-tg mice are therefore a new, promising model for studying EGFR-dependent gastric polyps.
Keywords: Epidermal growth factor receptor; Betacellulin; Gastric polyps; Mouse models;

Prolongation of carrageenan-induced inflammation in human colonic epithelial cells by activation of an NFκB‐BCL10 loop by Alip Borthakur; Sumit Bhattacharyya; Arivarasu N. Anbazhagan; Anoop Kumar; Pradeep K. Dudeja; Joanne K. Tobacman (1300-1307).
Carrageenan, a sulfated polysaccharide that is widely used as a food additive, induces inflammatory responses in animal models and human cells. The carrageenan-induced inflammatory cascades involve toll-like receptor (TLR)4- and B-cell leukemia/lymphoma (BCL)10-dependent activation of NF-κB, leading to increased IL-8 production. Translocations involving BCL10 in the mucosa-associated lymphoid tissue (MALT) lymphomas are associated with constitutive activation of NF-κB. This report presents a mechanism by which carrageenan exposure leads to prolonged activation of both BCL10 and NF-κB in human colonic epithelial cells. Study findings demonstrate that nuclear RelA and RelB bind to an NF-κB binding motif in the BCL10 promoter in human colonic epithelial NCM460 and HT-29 cells. In vitro oligonucleotide binding assay, non-radioactive gel shift assay, and chromatin immunoprecipitation (ChIP) indicate binding of RelA and RelB to the BCL10 promoter. Prolonged inflammation follows activation of the BCL10-NFκB inflammatory loop in response to carrageenan, shown by increased BCL10, RelA, and IL-8 for 36 to 48 h and increased RelB for 24 h following withdrawal of carrageenan after 12 h. In contrast, exposure to dextran sulfate sodium, which does not cause inflammation through TLR4 and BCL10 in the colonic epithelial cells, did not provoke prolonged activation of inflammation. The carrageenan-enhanced BCL10 promoter activity was blocked by caffeic acid phenethyl ester (CAPE) and MB-132 which inhibit NF-κB activation. These results indicate that NF-κB binding to the BCL10 promoter can lead to prolonged activation of the carrageenan-induced inflammatory cascade by a transcriptional mechanism involving an NF-κB‐BCL10 loop.► Exposure to common food additive carrageenan initiates protracted inflammation. ► The presence of NF-κB binding site in the BCL10 promoter sets up inflammatory loop. ► Both RelA and RelB bind to the NF-κB binding site in the BCL10 promoter. ► In contrast to carrageenan, DSS exposure does not initiate prolonged inflammation.
Keywords: NF-kappaB; BCL10; Carrageenan; Inflammation; IL-8; DSS;

HSF4 is involved in DNA damage repair through regulation of Rad51 by Xiukun Cui; Jing Zhang; Rong Du; Lei Wang; Stephen Archacki; Yuexuan Zhang; Mingxiong Yuan; Tie Ke; Hui Li; Duanzhuo Li; Chang Li; David Wan-Cheng Li; Zhaohui Tang; Zhan Yin; Mugen Liu (1308-1315).
Heat shock factor protein 4 (HSF4) is expressed exclusively in the ocular lens and plays a critical role in the lens formation and differentiation. Mutations in the HSF4 gene lead to congenital and senile cataract. However, the molecular mechanisms causing this disease have not been well characterized. DNA damage in lens is a crucial risk factor in senile cataract formation, and its timely repair is essential for maintaining the lens' transparency. Our study firstly found evidence that HSF4 contributes to the repair of DNA strand breaks. Yet, this does not occur with cataract causative mutations in HSF4. We verify that DNA damage repair is mediated by the binding of HSF4 to a heat shock element in the Rad51 promoter, a gene which assists in the homologous recombination (HR) repair of DNA strand breaks. HSF4 up-regulates Rad51 expression while mutations in HSF4 fail, and DNA does not get repaired. Camptothecin, which interrupts the regulation of Rad51 by HSF4, also affects DNA damage repair. Additionally, with HSF4 knockdown in the lens of Zebrafish, DNA damage was observed and the protein level of Rad51 was significantly lower. Our study presents the first evidence demonstrating that HSF4 plays a role in DNA damage repair and may contribute a better understanding of congenital cataract formation.► HSF4 promotes DNA breaks repair through the regulation of Rad51 expression. ► HSF4 pathogenic mutations abolish the DNA damage repair ability of HSF4. ► Camptothecin inhibits the up-regulation of Rad51 levels by HSF4. ► Knock down of HSF4 in Zebrafish decrease Rad51 levels and DNA repair.
Keywords: Cataract; HSF4; DNA damage; Rad51; Camptothecin;

Anti-amyloidogenic effects of soybean isoflavones in vitro: Fluorescence spectroscopy demonstrating direct binding to Aβ monomers, oligomers and fibrils by Mie Hirohata; Kenjiro Ono; Jun-ichi Takasaki; Ryoichi Takahashi; Tokuhei Ikeda; Akiyoshi Morinaga; Masahito Yamada (1316-1324).
Alzheimer's disease is characterized by the presence of extracellular deposits of amyloid, primarily composed of the amyloid β-protein (Aβ). A growing body of evidence indicates that oligomeric forms of Aβ play a critical role in disease causation. Soybean isoflavones are flavonoids with an isoflavone backbone. Isoflavones have been reported to protect against Aβ-induced neurotoxicity in cultured cell systems, the molecular mechanisms remain unclear. Our previous studies demonstrated that red wine-related flavonoids with a flavone backbone are able to inhibit Aβ assembly and destabilize preformed Aβ aggregates. Here, we show that isoflavones, especially glycitein and genistein, have anti-fibrillization, anti-oligomerization and fibril-destabilizing effects on Aβ1–40 and Aβ1–42 in vitro at physiological pH and temperature, by using nucleation-dependent polymerization monitored by thioflavin T fluorescence, atomic force microscopy, electron microscopy, and photo-induced cross-linking of unmodified proteins followed by SDS-PAGE. Our three-dimensional fluorescence spectroscopic analyses demonstrated that glycitein interacted with Aβ monomers, oligomers and fibrils, indicating specific binding of glycitein to these Aβ species. Glycitein also interacted with different Aβ fragments (Aβ1–42, Aβ1–40, Aβ1–16 and Aβ25–35), exhibiting the highest fluorescence enhancement with Aβ25–35. We speculated that glycitein's anti-amyloidogenic properties are specifically mediated by its binding to Aβ monomers, oligomers and fibrils. Isoflavones may hold promise as a treatment option for preventative strategies targeting amyloid formation in Alzheimer's disease.► Isoflavones inhibited the fibrillization and oligomerization of amyloid β-proteins (Aβs). ► Isoflavones destabilized preformed amyloid β-protein (Aβ) fibrils. ► Glycitein bound to Aβ monomers, oligomers and fibrils, leading the anti-amyloidogenic effects. ► Glycitein bound to Aβ1–42, Aβ1–40, Aβ1–16 and Aβ25–35, having the most potent affinity for Aβ25–35.
Keywords: Alzheimer's disease; Amyloid β-protein oligomer; Fragment; Photo-induced cross-linking of unmodified proteins (PICUP); Glycitein; Three-dimensional fluorescence spectroscopy;