BBA - Molecular Basis of Disease (v.1862, #11)

Disruption of calpain reduces lipotoxicity-induced cardiac injury by preventing endoplasmic reticulum stress by Shengcun Li; Lulu Zhang; Rui Ni; Ting Cao; Dong Zheng; Sidong Xiong; Peter A. Greer; Guo-Chang Fan; Tianqing Peng (2023-2033).
Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4 weeks) were fed a high fat diet (HFD) or normal diet for 20 weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mice. Mechanistically, deletion of capns1 in HFD-fed mouse hearts and disruption of calpain with calpain inhibitor-III, silencing of capn1, or deletion of capns1 in palmitate-stimulated cardiomyocytes prevented endoplasmic reticulum stress, apoptosis, cleavage of caspase-12 and junctophilin-2, and pro-inflammatory cytokine expression. Pharmacological inhibition of endoplasmic reticulum stress diminished palmitate-induced apoptosis and pro-inflammatory cytokine expression in cardiomyocytes. In summary, disruption of calpain prevents lipotoxicity-induced apoptosis in cardiomyocytes and cardiac injury in mice fed a HFD. The role of calpain is mediated, at least partially, through endoplasmic reticulum stress. Thus, calpain/endoplasmic reticulum stress may represent a new mechanism and potential therapeutic targets for cardiac lipotoxicity.
Keywords: Calpain; Cardiomyocytes; Lipotoxicity; Endoplasmic reticulum stress;

Enhanced alpha-kinase 1 accelerates multiple early nephropathies in streptozotocin-induced hyperglycemic mice by Tzer-Min Kuo; Hui-Ting Hsu; Chia-Min Chung; Kun-Tu Yeh; Cheng-Tien Wu; Chi-Pin Lee; Shang-Lun Chiang; Chung-Ming Huang; Ying-Chin Ko (2034-2042).
Alpha-kinase 1 (ALPK1) is associated with chronic kidney disease (CKD), type 2 diabetes mellitus and gout. Elevated ALPK1 levels have been observed in the kidneys of patients with diabetes and the white blood cells of patients with gout. As renal injury is a common outcome of CKD, diabetes and gout, the aim of this study was to investigate the effect of ALPK1 in the development of renal injury in a hyperglycemic condition. Hyperglycemia was induced in wild-type and ALPK1 transgenic mice by an intraperitoneal injection of streptozotocin (STZ). Functional and histological examinations were performed after 3 weeks. STZ-treated ALPK1 transgenic mice exclusively showed arteriolar sclerosis and fibrous thickening of the Bowman's capsule in the kidney. This was accompanied by body weight loss, severe hyperglycemia, and low serum insulin levels. Renal renin and serum renin protein levels were higher in STZ-treated ALPK1 transgenic mice, whereas cGKII protein level was decreased by ALPK1 in human embryonic kidney 293 (HEK293) cells. ALPK1 up-regulated TGF-beta1 levels and transcription of fibrosis-related genes, including MMP-9, FIBRONECTIN, and TIMP1. MSU crystals increased ALPK1 transcription in cultured kidney cells. Finally, ALPK1 enhanced production of MSU crystals-induced IL-1beta in mice. Stimulation of soluble sodium urate induced IL-1beta and Alpk1 mRNA production in mice kidney. Taken together, these data show that an increase in ALPK1 results in accelerated fibrotic nephropathies, primarily through the enhancement of renin, TGF-beta1, and IL-1beta. Renal or blood ALPK1 levels are involved in the induction of fibrotic renal injury in an experimental model of hyperglycemia.
Keywords: ALPK1; Diabetes; Renal injury; Fibrosis; IL-1 beta; TGF-beta1;

Hepatic carboxylesterase 3 (Ces3/Tgh) is downregulated in the early stages of liver cancer development in the rat by Ariel D. Quiroga; María P. Ceballos; Juan P. Parody; Carla G. Comanzo; Florencia Lorenzetti; Gerardo B. Pisani; María T. Ronco; María de L. Alvarez; María C. Carrillo (2043-2053).
It is accepted that cancer development is associated with metabolic changes. Previously, we established a model of hepatic preneoplasia in which adult rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP) for 6 weeks until they develop altered hepatic foci (AHF). Here, we found that a whole metabolic shift occurs in order to favor cancer development. IP animals presented with increased plasma lipids due to increased VLDL secretion as well as increased liver lipid accretion due to stimulated transacetylase activity rather than lipogenesis, compared to control rats. We found that carboxylesterase 3/triacylglycerol hydrolase (Ces3/Tgh) presented with a perilobular distribution surrounding lipid droplets in normal livers. However, it is downregulated both at the protein and mRNA level in liver homogenates and is almost undetectable inside the AHF with no changes in the surrounding tissue. Ces3/Tgh expression is regulated by ω-3 fatty acids, thus, supplementation of diet with fish oil, allowed the restoration of Ces3/Tgh expression inside the foci and, more interestingly, led to the decrease in number and volume of the AHF. These studies show a preventive role of Ces3/Tgh in liver cancer development.
Keywords: Carboxylesterases; Liver preneoplasia; Hypertriglyceridemia; Polyunsaturated fatty acids; Rat; Lipid metabolism;

Bile acid receptor agonists INT747 and INT777 decrease oestrogen deficiency-related postmenopausal obesity and hepatic steatosis in mice by Monique C. de Oliveira; Eduardo H. Gilglioni; Bouke A. de Boer; Jurgen H. Runge; Dirk R. de Waart; Clairce L. Salgueiro; Emy L. Ishii-Iwamoto; Ronald P.J. Oude Elferink; Ingrid C. Gaemers (2054-2062).
Menopause is often followed by obesity and, related to this, non-alcoholic fatty liver disease (NAFLD). Two bile acid (BA) receptors, farnesoid X receptor (FXR) and G-protein-coupled receptor TGR5, have emerged as putative therapeutic targets for obesity and NAFLD.Aim of this study: to evaluate the efficacy of selective agonists INT747/obeticholic acid (FXR) and INT777 (TGR5) as novel treatments for the metabolic effects of oestrogen deficiency.Ovariectomized (OVX) or sham-operated (SHAM) mice were fed a high-fat diet (HFD) for 5 weeks. During the last 4 weeks two groups of OVX and SHAM mice received either INT747- or INT777-supplemented HFD.OVX mice had significantly higher bodyweight gain than SHAM mice, which was attenuated by INT747- or INT777-treatment. No significant changes in food intake or physical activity were found. OVX mice had significantly lower energy expenditure than SHAM mice; INT747- and INT777-treated OVX mice had intermediate energy expenditure. Liver triglyceride and cholesterol content was significantly increased in OVX compared to SHAM mice, which was normalized by INT747- or INT777-treatment. Significant changes in metabolic gene expression were found in liver (Cpt1, Acox1), muscle (Ucp3, Pdk4, Cpt1, Acox1, Fasn, Fgf21), brown adipocytes (Dio2) and white adipocytes (c/EBPα, Pparγ, Adipoq). For the first time, expression of FXR and induction of its target gene Pltp1 was shown in skeletal muscle.BA receptor agonists are suitable therapeutics to correct postmenopausal metabolic changes in an OVX mouse model. Potential mechanisms include increased energy expenditure and changes in expression patterns of key metabolic genes in liver, muscle and adipose tissues.
Keywords: Ovariectomy; Nonalcoholic fatty liver disease; Steatosis; Bile acid receptor FXR agonist INT-747; Bile acid receptor TGR5 agonist INT-777; Obeticholic acid;

Higher susceptibility of cerebral cortex and striatum to sulfite neurotoxicity in sulfite oxidase-deficient rats by Mateus Grings; Alana Pimentel Moura; Belisa Parmeggiani; Marcela Moreira Motta; Rafael Mello Boldrini; Pauline Maciel August; Cristiane Matté; Angela T.S. Wyse; Moacir Wajner; Guilhian Leipnitz (2063-2074).
Patients affected by sulfite oxidase (SO) deficiency present severe seizures early in infancy and progressive neurological damage, as well as tissue accumulation of sulfite, thiosulfate and S-sulfocysteine. Since the pathomechanisms involved in the neuropathology of SO deficiency are still poorly established, we evaluated the effects of sulfite on redox homeostasis and bioenergetics in cerebral cortex, striatum, cerebellum and hippocampus of rats with chemically induced SO deficiency. The deficiency was induced in 21-day-old rats by adding 200 ppm of tungsten, a molybdenum competitor, in their drinking water for 9 weeks. Sulfite (70 mg/kg/day) was also administered through the drinking water from the third week of tungsten supplementation until the end of the treatment. Sulfite decreased reduced glutathione concentrations and the activities of glutathione reductase and glutathione S-transferase (GST) in cerebral cortex and of GST in cerebellum of SO-deficient rats. Moreover, sulfite increased the activities of complexes II and II-III in striatum and of complex II in hippocampus, but reduced the activity of complex IV in striatum of SO-deficient rats. Sulfite also decreased the mitochondrial membrane potential in cerebral cortex and striatum, whereas it had no effect on mitochondrial mass in any encephalic tissue evaluated. Finally, sulfite inhibited the activities of malate and glutamate dehydrogenase in cerebral cortex of SO-deficient rats. Taken together, our findings indicate that cerebral cortex and striatum are more vulnerable to sulfite-induced toxicity than cerebellum and hippocampus. It is presumed that these pathomechanisms may contribute to the pathophysiology of neurological damage found in patients affected by SO deficiency.
Keywords: Sulfite oxidase deficiency; Sulfite; Antioxidant defenses; Energy metabolism; Brain;

Signaling in dopamine D2 receptor-oxytocin receptor heterocomplexes and its relevance for the anxiolytic effects of dopamine and oxytocin interactions in the amygdala of the rat by Miguel Pérez de la Mora; Diana Pérez-Carrera; Minerva Crespo-Ramírez; Alexander Tarakanov; Kjell Fuxe; Dasiel O. Borroto-Escuela (2075-2085).
Dopamine D2 receptor (D2R)–oxytocin receptor (OTR) interactions exist within heterocomplexes with facilitatory effects on D2R recognition and Gi/o coupling. In this work the hypothesis is tested using cotransfected HEK293 cells whether allosteric reciprocal D2R-OTR interactions can enhance signaling of D2R-OTR heterocomplexes along the CREB, MAPK and PLC pathways and whether the anxiolytic effects of OT may involve facilitatory D2R-OTR interactions within the central amygdaloid nucleus (CeA). Oxytocin enhanced the D2-like agonist quinpirole induced inhibition of the AC-PKA-pCREB signaling cascade and increased its signaling over the RAS-MAPK-pELK pathway. Quinpirole enhanced the oxytocin induced increases in the activity of the PLCbeta-IP3-calcineurin and RAS-MAPK-pELK cascades. Bilateral infusion of oxytocin (0.9–150 ng/side) into the CeA of the rat elicited anxiolytic effects in the Shock-Probe Burying test, an unconditioned model of fear/anxiety. This action was not observed when oxytocin (25 ng/side) was simultaneously co-infused with raclopride (neither 250 nor 500 ng/side), a D2/D3 antagonist, into the CeA. Based on the current findings, the blockade of the anxiolytic effects of oxytocin by the simultaneous intra-CeA administration of raclopride can be explained by a lack of facilitatory protomer interactions in D2R-OTR heterocomplexes. Dysfunction and/or disruption of such interactions in the central amygdala may lead to anxiety development. Restoration of such interactions may represent a new strategy for development of novel anxiolytic drugs.
Keywords: Anxiety; Fear; Amygdala; Dopamine D2 receptor; Oxytocin receptor; Receptor-receptor interactions; Raclopride; Oxytocin; Heteroreceptor complexes;

Modulation of hepatic copper-ATPase activity by insulin and glucagon involves protein kinase A (PKA) signaling pathway by Elaine Hilário-Souza; Martine Cuillel; Elisabeth Mintz; Peggy Charbonnier; Adalberto Vieyra; Doris Cassio; Jennifer Lowe (2086-2097).
Different studies have revealed copper imbalance in individuals suffering from diabetes and obesity, suggesting that regulation of glucose and/or fat metabolism could modulate cellular copper homeostasis. To test this hypothesis we investigated whether the key hormones of energy metabolism, insulin and glucagon, regulate the functional properties of the major hepatic copper-transporter, ATP7B (i.e., copper-dependent ATPase activity). We demonstrated that insulin reverses the effect of copper and stimulates retrograde trafficking of ATP7B from the canalicular membranes, consistent with the enhanced ability of ATP7B to sequester copper away from the cytosol. Physiological concentrations of insulin increase endogenous ATP7B activity in cultured hepatic cells and in tissues by 40%, whereas glucagon inhibits this activity by 70%. These effects were cancelled out when insulin and glucagon were combined. We also demonstrated that the opposite effects of the hormones on ATP7B activity involve receptor-mediated signaling pathways and membrane-bound kinases (PKA and PKB/Akt), which are reciprocally regulated by insulin and glucagon. Inhibiting insulin signaling at the level of its Tyr-kinase receptor, PI3K or PKB/Akt restored the basal activity of ATP7B. Insulin reduced endogenous PKA activity, whereas glucagon promoted PKA stimulation by approximately 100%. These findings demonstrate that the physiological modulation of ATP7B activity is linked to energy metabolism via insulin and glucagon, and could help to understand the mechanisms involved in the disruption of copper homeostasis in diabetic and obese patients.
Keywords: Copper imbalance; Copper transport; Glucagon; Insulin; Protein kinase A (PKA);

Pathogenic amino acid substitutions of the common E3 component (hE3) of the human alpha-ketoglutarate dehydrogenase and the pyruvate dehydrogenase complexes lead to severe metabolic diseases (E3 deficiency), which usually manifest themselves in cardiological and/or neurological symptoms and often cause premature death. To date, 14 disease-causing amino acid substitutions of the hE3 component have been reported in the clinical literature. None of the pathogenic protein variants has lent itself to high-resolution structure elucidation by X-ray or NMR. Hence, the structural alterations of the hE3 protein caused by the disease-causing mutations and leading to dysfunction, including the enhanced generation of reactive oxygen species by selected disease-causing variants, could only be speculated. Here we report results of an examination of the effects on the protein structure of ten pathogenic mutations of hE3 using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), a new and state-of-the-art approach of solution structure elucidation. On the basis of the results, putative structural and mechanistic conclusions were drawn regarding the molecular pathogenesis of each disease-causing hE3 mutation addressed in this study.Display Omitted
Keywords: Dihydrolipoamide dehydrogenase; Pathogenic mutation; E3 deficiency; Hydrogen/deuterium exchange; Mass spectrometry;

Anthocyanins protect from complex I inhibition and APPswe mutation through modulation of the mitochondrial fission/fusion pathways by Cristina Parrado-Fernández; Anna Sandebring-Matton; Patricia Rodriguez-Rodriguez; Dag Aarsland; Angel Cedazo-Mínguez (2110-2118).
Anthocyanins are a distinguished class of flavonoids with powerful free radical-scavenging activity that have been suggested as chemotherapeutic agents for the prevention of Alzheimer disease (AD). In this study, we examined the ability of nutraceutical Medox rich in purified cyanidin 3-O-glucoside (C3G), 3-O-b-glucosides and delphinidin 3-O-glucoside (D3G) to counteract mitochondrial deficiency induced by complex I inhibition and/or amyloid-β peptide (Aβ) induced toxicity. SH-SY5Y neuroblastoma cells were stably transfected with APP Swedish K670N/M671L double mutation (APPswe) or with the empty vector and treated with rotenone. We report that Medox treatment improves the metabolic activity and maintains cell integrity in both cell lines. At the mitochondrial level, APPswe and rotenone induced mitochondrial fragmentation, an effect that was counteracted by Medox through the modulation of fission and fusion proteins, resulting in a reshaped mitochondrial network. Although Medox was unable to fully neutralise the effects of rotenone on ATP levels and mitochondrial membrane potential, it was able to prevent rotenone-induced cytotoxicity.Our findings suggest that Medox anthocyanins, on top of their antioxidant capacity, ameliorate mitochondrial dysfunction generated by Aβ overproduction or by chemical inhibition of mitochondrial complex I via stabilization of the fusion/fission processes. Modulation of the mitochondrial network has been suggested as a novel therapeutic approach in diseases involving mitochondrial dysfunction and oxidative stress. Hence, increasing the understanding of how anthocyanins influence mitochondrial dynamics in a neurodegenerative context, could be of future therapeutic value.
Keywords: Anthocyanins; Mitochondrial dynamics; Beta-amyloid; Rotenone; Neuroprotection;

miR-431 is involved in regulating cochlear function by targeting Eya4 by Yue Fan; Ying Zhang; Rimao Wu; Xingming Chen; Yong Zhang; Xiaowei Chen; Dahai Zhu (2119-2126).
To understand the relationship between microRNAs and hearing loss and help clarify the causes of hereditary deafness, we studied the functions of miR-431 in cochleae. We first investigated the spatial-temporal expression profiles of miR-431 in spiral ganglion neurons (SGNs) in cochleae using real-time PCR and miRNA in situ hybridization. These studies showed that expression of miR-431 was high in SGNs in the cochleae of newborn mice, and decreased as development progressed. To test the functional effects of miR-431, we established miR-431 overexpressing transgenic (Tg) mice. Surface preparations of the cochlear basilar membrane and cochlear sections revealed no major structural differences between Tg and wild-type (Wt) mice. However, a comparison of auditory brain stem responses (ABRs) in Tg and Wt mice showed that ABR thresholds were significantly higher in Tg mice than in Wt mice. Notably, the density of SGNs was significantly lower in Tg mice than in Wt mice. We also found that the proportion of mature SGNs in cultures of primary SGNs from Tg cochleae was lower and their axons were shorter. A bioinformatics analysis predicted that the mRNA target of miR-431 was Eya4, a finding confirmed by luciferase reporter assays and western blotting. Importantly, overexpression of miR-431 in cochleae of Tg mice inhibited the translation of Eya4 mRNA, leading to a deficiency of EYA4. Thus, excessive amounts of miR-431 in cochleae of Tg mice may be the cause of sparse SGNs, which in turn could be responsible for hearing loss.
Keywords: miR-431; Eya4; Transgenic mouse; Cochleae; Spiral ganglion neuron;

Negative feedback circuitry between MIR143HG and RBM24 in Hirschsprung disease by Chunxia Du; Ziyang Shen; Rujin Zang; Hua Xie; Hongxing Li; Pingfa Chen; Bo Hang; Xiaoqun Xu; Weibing Tang; Yankai Xia (2127-2136).
Hirschsprung disease (HSCR) is a genetic disorder of neural crest development. It is also believed that epigenetic changes plays a role in the progression of this disease. Here we show that the MIR143 host gene (MIR143HG), the precursor of miR-143 and miR-145, decreased cell proliferation and migration and forms a negative feedback loop with RBM24 in HSCR. As RBM24 mRNA is a target of miR-143, upregulation of RBM24 upon an increase in the level of MIR143HG could be attributed to sequestration of miR-143 by MIR143HG (sponge effect). The RBM24 protein was shown to bind to MIR143HG, and subsequently, accelerated its degradation by destabilizing its transcript and facilitating its interaction with Ago2, thus forming a negative feedback between MIR143HG and RBM24. In addition, experiments using siRNA against DROSHA indicated that RBM24 could promote the biogenesis of miR-143. This feedback loop we describe here represents a novel mode of autoregulation, with implications in HSCR pathogenesis.
Keywords: Hirschsprung disease; Neuronal development; Long non-coding RNA (lncRNA); Competing endogenous RNA (CeRNA); MiR-143;

In utero exposure to gestational diabetes mellitus conditions TLR4 and TLR2 activated IL-1beta responses in spleen cells from rat offspring by Qian Li; Troy J. Pereira; Brittany L. Moyce; Thomas H. Mahood; Christine A. Doucette; Julia Rempel; Vernon W. Dolinsky (2137-2146).
Fetal exposure to gestational diabetes mellitus (GDM) is associated with a higher risk of youth-onset insulin resistance and type 2 diabetes. We have previously shown that the rat offspring of GDM dams are insulin resistant when compared to the offspring of lean dams. Since inflammation influences insulin sensitivity, we examined the impact of fetal exposure to GDM on inflammatory responses in the offspring. In rats, we compared inflammatory activity in newborn pups as well as 16 week-old young-adult offspring from lean control dams with offspring from high fat and sucrose diet (HFS)-induced GDM dams. To determine whether there are additive effects of exposure to GDM and post-weaning diets, offspring of lean and GDM dams were fed either low fat or HFS diets until 16 weeks of age. Plasma levels of interleukin(IL)-1β were elevated in the offspring of GDM dams. To determine whether this was related to immune reactivity, spleen cells from both the newborn and 16 week-old offspring were isolated and reactivity to the toll-like receptor activators, pam3CSK4 and lipopolysaccharides were measured over a 72 h timeframe. Spleen cells of GDM dams exhibited sustained stimulation of interleukin(IL)-1β and IL-10 production, whereas IL-1β and IL-10 synthesis diminished over time in spleen cells from the offspring of lean dams. Additive effects of GDM exposure and post-weaning HFS diet were not observed, suggesting the effects of GDM on cytokine production are independent of the post-weaning diet. Thus, we conclude that exposure to GDM in utero may condition the immune reactivity of spleen cells.
Keywords: Inflammation; Fetal programming; Gestational diabetes;

An impaired metabolism of nucleotides underpins a novel mechanism of cardiac remodeling leading to Huntington's disease related cardiomyopathy by Marta Toczek; Daniel Zielonka; Paulina Zukowska; Jerzy T. Marcinkowski; Ewa Slominska; Mark Isalan; Ryszard T. Smolenski; Michal Mielcarek (2147-2157).
Huntington's disease (HD) is mainly thought of as a neurological disease, but multiple epidemiological studies have demonstrated a number of cardiovascular events leading to heart failure in HD patients. Our recent studies showed an increased risk of heart contractile dysfunction and dilated cardiomyopathy in HD pre-clinical models. This could potentially involve metabolic remodeling, that is a typical feature of the failing heart, with reduced activities of high energy phosphate generating pathways. In this study, we sought to identify metabolic abnormalities leading to HD-related cardiomyopathy in pre-clinical and clinical settings. We found that HD mouse models developed a profound deterioration in cardiac energy equilibrium, despite AMP-activated protein kinase hyperphosphorylation. This was accompanied by a reduced glucose usage and a significant deregulation of genes involved in de novo purine biosynthesis, in conversion of adenine nucleotides, and in adenosine metabolism. Consequently, we observed increased levels of nucleotide catabolites such as inosine, hypoxanthine, xanthine and uric acid, in murine and human HD serum. These effects may be caused locally by mutant HTT, via gain or loss of function effects, or distally by a lack of trophic signals from central nerve stimulation. Either may lead to energy equilibrium imbalances in cardiac cells, with activation of nucleotide catabolism plus an inhibition of re-synthesis. Our study suggests that future therapies should target cardiac mitochondrial dysfunction to ameliorate energetic dysfunction. Importantly, we describe the first set of biomarkers related to heart and skeletal muscle dysfunction in both pre-clinical and clinical HD settings.
Keywords: Huntington's disease; Cardiomyopathy; Arrhythmia; Energy imbalance; Catabolism of nucleotides; Heart failure;

c-Abl links APP-BACE1 interaction promoting APP amyloidogenic processing in Niemann-Pick type C disease by M.J. Yáñez; O. Belbin; L.D. Estrada; N. Leal; P.S. Contreras; A. Lleó; P.V. Burgos; S. Zanlungo; A.R. Alvarez (2158-2167).
Niemann-Pick type C (NPC) disease is characterized by lysosomal accumulation of cholesterol. Interestingly, NPC patients' brains also show increased levels of amyloid-β (Aβ) peptide, a key protein in Alzheimer's disease pathogenesis.We previously reported that the c-Abl tyrosine kinase is active in NPC neurons and in AD animal models and that Imatinib, a specific c-Abl inhibitor, decreased the amyloid burden in brains of the AD mouse model. Active c-Abl was shown to interact with the APP cytosolic domain, but the relevance of this interaction to APP processing has yet to be defined.In this work we show that c-Abl inhibition reduces APP amyloidogenic cleavage in NPC cells overexpressing APP. Indeed, we found that levels of the Aβ oligomers and the carboxy-terminal fragment βCTF were decreased when the cells were treated with Imatinib and upon shRNA-mediated c-Abl knockdown. Moreover, Imatinib decreased APP amyloidogenic processing in the brain of an NPC mouse model. In addition, we found decreased levels of βCTF in neuronal cultures from c-Abl null mice. We demonstrate that c-Abl directly interacts with APP, that c-Abl inhibition prevents this interaction, and that Tyr682 in the APP cytoplasmic tail is essential for this interaction. More importantly, we found that c-Abl inhibition by Imatinib significantly inhibits the interaction between APP and BACE1.We conclude that c-Abl activity facilitates the APP-BACE1 interaction, thereby promoting amyloidogenic processing of APP. Thus, inhibition of c-Abl could be a pharmacological target for preventing the injurious effects of increased Aβ levels in NPC disease.
Keywords: Niemann-Pick type C; Amyloid beta; Amyloid precursor protein; β-secretase; Tyrosine kinase c-Abl; Imatinib;

Connexin hemichannels explain the ionic imbalance and lead to atrophy in denervated skeletal muscles by Bruno A. Cisterna; Aníbal A. Vargas; Carlos Puebla; Juan C. Sáez (2168-2176).
Denervated fast skeletal muscles undergo atrophy, which is associated with an increase in sarcolemma permeability and protein imbalance. However, the mechanisms responsible for these alterations remain largely unknown. Recently, a close association between de novo expression of hemichannels formed by connexins 43 and 45 and increase in sarcolemma permeability of denervated fast skeletal myofibers was demonstrated. However, it remains unknown whether these connexins cause the ionic imbalance of denervates fast myofibers. To elucidate the latter and the role of hemichannels formed by connexins (Cx HCs) in denervation-induced atrophy, skeletal myofibers deficient in Cx43 and Cx45 expression (Cx43fl/flCx45fl/fl:Myo-Cre mice) and control (Cx43fl/flCx45fl/fl mice) were denervated and several muscle features were systematically analyzed at different post-denervation (PD) times (1, 3, 5, 7 and 14 days). The following sequence of events was found in denervated myofibers of Cx43fl/flCx45fl/fl mice: 1) from day 3 PD, increase in sarcolemmal permeability, 2) from day 5 PD, increases of intracellular Ca2+ and Na+ signals as well as a significant increase in protein synthesis and degradation, yielding a negative protein balance and 3) from day 7 PD, a fall in myofibers cross-section area. All the above alterations were either absent or drastically reduced in denervated myofibers of Cx43fl/flCx45fl/fl:Myo-Cre mice. Thus, the denervation-induced Cx HCs expression is an early event that precedes the electrochemical gradient dysregulation across the sarcolemma and critically contributes to the progression of skeletal muscle atrophy. Consequently, Cx HCs could be a therapeutic target to drastically prevent the denervation-induced atrophy of fast skeletal muscles.
Keywords: Calcium ion; Sodium ion; Protein synthesis; Protein degradation; Skeletal muscle atrophy;

Increasing evidence has shown that microRNAs played an important role in regulating carcinogenesis. However, the role of miR-29a in breast cancer is still unclear. Herein, we showed that miR-29a was significantly up-regulated in breast cancer as compared with non-tumor tissues. Moreover, the up-regulation of miR-29a was significantly correlated with tumor metastasis and shorter overall survival in breast cancer patients. Knockdown of miR-29a in breast cancer cell lines inhibited cell proliferation and migration. Furthermore, data from bioinformatic analysis validated by dual-luciferase reporter gene assay showed that ten eleven translocation 1 (TET1) was a direct target of miR-29a, and over-expression of TET1 inhibited cell proliferation and migration which could be induced by the up-regulation of miR-29a. TET1 silencing promoted cell growth and migration in breast cancer. MiR-29a over-expression had the same effect. MiR-29a targets TET1, down regulates its expression and thus promotes EMT in breast cancer. Altogether, we demonstrate that miR-29a acts as a tumor activator by targeting TET1 and induces cell proliferation and EMT in breast cancer.
Keywords: Breast cancer; Cell proliferation; EMT; MiR-29a; TET1;

Sex-determining region Y-box 9 acts downstream of NADPH oxidase to influence the effect of leptin on PPARγ1 expression in hepatic stellate cells by Haowen Qiao; Qing Cao; Yucheng Fu; Wei Guan; Fangyun Cheng; Juanjuan Wu; Xin Jia; Hongshan Chen; Yajun Zhou (2186-2196).
Leptin, an adipocyte-derived hormone, promotes liver fibrogenesis and inhibits the expression of peroxisome-proliferator activated receptor γ (PPARγ), a key transcription factor in inhibition of hepatic stellate cell (HSC) activation, in HSCs. This research aimed to further investigate the mechanisms underlying leptin regulation of PPARγ1 in HSCs in vivo and in vitro. Results demonstrated that sex-determining region Y-box 9 (Sox9) could bind to a site around − 2275 within leptin response region of PPARγ1 promoter and inhibited PPARγ1 expression. Sox9 upregulated the expressions of α1(I)collagen and alpha-smooth muscle actin in HSCs. Leptin stimulated Sox9 expression and Sox9 binding to PPARγ1 promoter. The signaling pathways of NADPH oxidase, β-catenin, and delta-like homolog1 (DLK1) mediated leptin upregulation of Sox9 expression. Moreover, there existed crosstalk between NADPH oxidase pathway and β-catenin or DLK1 signaling pathway. Human liver specimens of cirrhosis were shown to be of a large number of the positive HSCs for p47phox (playing a central role in NADPH oxidase activity), 4-hydroxynonenal (a lipid peroxidation product), Sox9, and α-smooth muscle actin whereas PPARγ-positive HSCs were rarely detected. These results might deepen understanding of the molecular mechanisms for leptin inhibition of PPARγ1 expression in HSCs and for the liver fibrosis associated with leptin.Display Omitted
Keywords: Leptin; Sex-determining region Y-box 9; Peroxisome-proliferator activated receptor γ1; NADPH oxidase; Hepatic stellate cell; Liver fibrosis;