BBA - Molecular Basis of Disease (v.1832, #8)

V-ATPase is a candidate therapeutic target for Ewing sarcoma by Sofia Avnet; Gemma Di Pompo; Silvia Lemma; Manuela Salerno; Francesca Perut; Gloria Bonuccelli; Donatella Granchi; Nicoletta Zini; Nicola Baldini (1105-1116).
Suppression of oxidative phosphorylation combined with enhanced aerobic glycolysis and the resulting increased generation of protons are common features of several types of cancer. An efficient mechanism to escape cell death resulting from intracellular acidification is proton pump activation. In Ewing sarcoma (ES), although the tumor-associated chimeric gene EWS-FLI1 is known to induce the accumulation of hypoxia-induced transcription factor HIF-1α, derangements in metabolic pathways have been neglected so far as candidate pathogenetic mechanisms. In this paper, we observed that ES cells simultaneously activate mitochondrial respiration and high levels of glycolysis. Moreover, although the most effective detoxification mechanism of proton intracellular storage is lysosomal compartmentalization, ES cells show a poorly represented lysosomal compartment, but a high sensitivity to the anti-lysosomal agent bafilomycin A1, targeting the V-ATPase proton pump. We therefore investigated the role of V-ATPase in the acidification activity of ES cells. ES cells with the highest GAPDH and V-ATPase expression also showed the highest acidification rate. Moreover, the localization of V-ATPase was both on the vacuolar and the plasma membrane of all ES cell lines. The acidic extracellular pH that we reproduced in vitro promoted high invasion ability and clonogenic efficiency. Finally, targeting V-ATPase with siRNA and omeprazole treatments, we obtained a significant selective reduction of tumor cell number. In summary, glycolytic activity and activation of V-ATPase are crucial mechanisms of survival of ES cells and can be considered as promising selective targets for the treatment of this tumor.
Keywords: Ewing sarcoma; Glycolysis; Extracellular acidification; V-ATPase;

Repression of adipogenesis through promotion of Wnt/β-catenin signaling by TIS7 up-regulated in adipocytes under hypoxia by Yukari Nakamura; Eiichi Hinoi; Takashi Iezaki; Saya Takada; Syota Hashizume; Yoshifumi Takahata; Emiko Tsuruta; Satoshi Takahashi; Yukio Yoneda (1117-1128).
Although tetradecanoyl phorbol acetate induced sequence-7 (TIS7) has been identified as a co-activator/repressor of gene transcription in different eukaryotic cells, little attention has been paid to the functionality of TIS7 in adipocytes. Here, we evaluated the possible role of TIS7 in mechanisms underlying the regulation of adipogenesis. TIS7 expression was preferentially up-regulated in white adipose tissues (WAT) of obesity model mice as well as in pre-adipocytic 3T3-L1 cells cultured under hypoxic conditions. TIS7 promoter activity was selectively enhanced by activating transcription factor-6 (ATF6) among different transcription factors tested, while induction of TIS7 by hypoxic stress was markedly prevented by knockdown of ATF6 by shRNA in 3T3-L1 cells. Overexpression of TIS7 markedly inhibited Oil Red O staining and expression of particular adipogenic genes in 3T3-L1 cells. TIS7 synergistically promoted gene transactivation mediated by Wingless-type mouse mammary tumor virus integration site family (Wnt)/β-catenin, while blockade of the Wnt/β-catenin pathway by a dominant negative form of T-cell factor-4 (DN-TCF4) markedly prevented the inhibition of adipogenesis in 3T3-L1 cells with TIS7 overexpression. TIS7 predominantly interacted with β-catenin in the nucleus of WAT in the genetically obese ob/ob mice as well as in 3T3-L1 cells cultured under hypoxic conditions. Both knockdown of TIS7 by shRNA and introduction of DN-TCF4 similarly reversed the hypoxia-induced inhibition of adipogenic gene expression in 3T3-L1 cells. These findings suggest that TIS7 could play a pivotal role in adipogenesis through interacting with β-catenin to promote the canonical Wnt signaling in pre-adipocytes under hypoxic stress such as obesity.
Keywords: TIS7; Adipogenesis; Wnt signaling; Hypoxia; ATF6;

Genome wide array analysis indicates that an amyotrophic lateral sclerosis mutation of FUS causes an early increase of CAMK2N2 in vitro by Paolo Convertini; Jiayu Zhang; Pierre de la Grange; Lawrence J. Hayward; Haining Zhu; Stefan Stamm (1129-1135).
Mutations in the RNA binding protein FUS (fused in sarcoma) have been linked to a subset of familial amyotrophic lateral sclerosis (ALS) cases. The mutations are clustered in the C-terminal nuclear localization sequence (NLS). Various FUS mutants accumulate in the cytoplasm whereas wild-type (WT) FUS is mainly nuclear. Here we investigate the effect of one ALS causing mutant (FUS-ΔNLS, also known as R495X) on pre-mRNA splicing and RNA expression using genome wide exon-junction arrays. Using a non-neuronal stable cell line with inducible FUS expression, we detected early changes in RNA composition. In particular, mutant FUS-ΔNLS increased calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2) at both mRNA and protein levels, whereas WT-FUS had no effect. Chromatin immunoprecipitation experiments showed that FUS-ΔNLS accumulated at the CAMK2N2 promoter region, whereas promoter occupation by WT-FUS remained constant. Given the loss of FUS-ΔNLS in the nucleus through the mutation-induced translocation, this increase of promoter occupancy is surprising. It indicates that, despite the obvious cytoplasmic accumulation, FUS-ΔNLS can act through a nuclear gain of function mechanism.
Keywords: FUS; Fused in sarcoma; Amyotrophic lateral sclerosis; ALS; Array analysis;

Adiponectin (APN), an adipose-derived adipokine, offers cardioprotective effects although the precise mechanism of action remains unclear. This study was designed to examine the role of APN in high fat diet-induced obesity and cardiac pathology. Adult C57BL/6 wild-type and APN knockout mice were fed a low or high fat diet for 22 weeks. After 40 day feeding, mice were treated with 2 mg/kg rapamycin or vehicle every other day for 42 days on respective fat diet. Cardiomyocyte contractile and Ca2 + transient properties were evaluated. Myocardial function was evaluated using echocardiography. Dual energy X-ray absorptiometry was used to evaluate adiposity. Energy expenditure, metabolic rate and physical activity were monitored using a metabolic cage. Lipid deposition, serum triglyceride, glucose tolerance, markers of autophagy and fatty acid metabolism including LC3, p62, Beclin-1, AMPK, mTOR, fatty acid synthase (FAS) were evaluated. High fat diet intake induced obesity, systemic glucose intolerance, cardiac hypertrophy, dampened metabolic ability, cardiac and intracellular Ca2 + derangements, the effects of which were accentuated by APN knockout. Furthermore, APN deficiency augmented high fat diet-induced upregulation in the autophagy adaptor p62 and the decline in AMPK without affecting high fat diet-induced decrease in LC3II and LC3II-to-LC3I ratio. Neither high fat diet nor APN deficiency altered Beclin-1. Interestingly, rapamycin negated high fat diet-induced/APN-deficiency-accentuated obesity, cardiac hypertrophy and contractile dysfunction as well as AMPK dephosphorylation, mTOR phosphorylation and p62 buildup. Our results collectively revealed that APN deficiency may aggravate high fat diet-induced obesity, metabolic derangement, cardiac hypertrophy and contractile dysfunction possibly through decreased myocardial autophagy.
Keywords: Adiponectin; Autophagy; Cardiac function; Hypertrophy; Metabolism; Obesity;

Hyperhomocysteinemia is detrimental to pregnancy in mice and is associated with preterm birth by S.R. Sonne; V.K. Bhalla; S.A. Barman; R.E. White; S. Zhu; T.M. Newman; P.D. Prasad; S.B. Smith; S. Offermanns; V. Ganapathy (1149-1158).
Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine β-synthase (Cbs+/− ). Gestational period was monitored in wild type and Cbs+/− female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BKCa channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE2 production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BKCa channel activity in human myometrial cells. Deletion of Gpr109a in Cbs+/− mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE2 axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth.
Keywords: Preterm birth; Homocysteine; Niacin receptor; Cyclooxygenase-2; Prostaglandin E2;

Intermittent-hypoxia induced autophagy attenuates contractile dysfunction and myocardial injury in rat heart by Hideyuki Maeda; Hisashi Nagai; Genzou Takemura; Kaori Shintani-Ishida; Masaaki Komatsu; Sayoko Ogura; Toshihiko Aki; Mikiayasu Shirai; Ichiro Kuwahira; Ken-ichi Yoshida (1159-1166).
Sleep apnea syndrome (SAS) is considered to be associated with heart failure (HF). It is known that autophagy is induced in various heart diseases thereby promotes survival, but its excess may be maladaptive. Intermittent hypoxia (IH) plays pivotal role in the pathogenesis of SAS. We aimed to clarify the relationships among IH, autophagy, and HF. Rats underwent IH at a rate of 20 cycles/h (nadir of 4% O2 to peak of 21% O2 with 0% CO2) or normal air breathing (control) for 8 h/d for 3 weeks. IH increased the cardiac LC3II/LC3I ratio. The IH induced upregulation of LC3II was attenuated by the administration of an inhibitor of autophagosome formation 3-methyladenine (3-MA), but enhanced by an inhibitor of autophagosome–lysosome fusion chloroquine (CQ), showing enhanced autophagic flux in IH hearts. Electron microscopy confirmed an increase in autophagosomes and lysosomes in IH. With 3-MA or CQ, IH induced progressive deterioration of fractional shortening (FS) on echocardiography over 3 weeks, although IH, 3-MA, or CQ alone had no effects. With CQ, IH for 4 weeks increased serum troponin T levels, reflecting necrosis. Western blotting analyses showed dephosphorylation of Akt and mammalian target of rapamycin (mTOR) at Akt (Ser2448, 2481) sites, suggesting the activation of autophagy via Akt inactivation. Conclusions. IH-mediated autophagy maintains contractile function, whereas when autophagy is inhibited, IH induces systolic dysfunction due to myocyte necrosis. General significance. This study highlighted the potential implications of autophagy in cardio-protection in early SAS patients without comorbidity, reproduced in normal rats by 3 ~ 4 weeks of IH.
Keywords: Sleep apnea syndrome; Heart failure; Autophagy; Intermittent hypoxia;

HSF4 regulates DLAD expression and promotes lens de-nucleation by Xiukun Cui; Lei Wang; Jing Zhang; Rong Du; Shengjie Liao; Duanzhuo Li; Chang Li; Tie Ke; David Wan-Cheng Li; Hua Huang; Zhan Yin; Zhaohui Tang; Mugen Liu (1167-1172).
Keywords: HSF4; Cataract mutation; DLAD; Lens differentiation;

Caprin-1, a novel Cyr61-interacting protein, promotes osteosarcoma tumor growth and lung metastasis in mice by Adam A. Sabile; Matthias J.E. Arlt; Roman Muff; Knut Husmann; Daniel Hess; Josefine Bertz; Bettina Langsam; Caroline Aemisegger; Urs Ziegler; Walter Born; Bruno Fuchs (1173-1182).
Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents. More than 30% of patients develop lung metastasis, which is the leading cause of mortality. Recently, the extracellular matrix protein Cyr61 has been recognized as a malignancy promoting protein in OS mouse model with prognostic potential in human OS. In this study, we aimed at the identification of novel Cyr61-interacting proteins.Here we report that Cyr61 associates with Caprin-1, and confocal microscopy showed that stable ectopic expression of Caprin-1 leads to the formation of stress granules containing Caprin-1 and Cyr61, confers resistance to cisplatin-induced apoptosis, and resulted in constitutive phosphorylation of Akt and ERK1/2. Importantly, ectopic expression of Caprin-1 dramatically enhanced primary tumor growth, remarkably increased lung metastatic load in a SCID intratibial OS mouse model, and decreased significantly (p  < 0.0018) the survival of the mice. Although Caprin-1 expression, evaluated with a tissue microarray including samples from 59 OS patients, failed to be an independent predictor for the patients' outcome in this limited cohort of patients, increased Caprin-1 expression indicated a tendency to shortened overall survival, and more strikingly, Cyr61/Caprin-1 co-expression was associated with worse survival than that observed for patients with tumors expressing either Cyr61 or Caprin-1 alone or none of these proteins. The findings imply that Caprin-1 may have a metastasis promoting role in OS and show that through resistance to apoptosis and via the activation of Akt and ERK1/2 pathways, Caprin-1 is significantly involved in the development of OS metastasis.
Keywords: Osteosarcoma; Metastasis; Cyr61; Caprin-1; Stress granules; Orthotopic mouse model;

Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.
Keywords: Cellular models; Intracellular aggregation; Leptomeningeal amyloidosis; Lysosomes; N-glycosylation; Transthyretin;

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation by François Gonzalvez; Marilena D'Aurelio; Marie Boutant; Aoula Moustapha; Jean-Philippe Puech; Thomas Landes; Laeticia Arnauné-Pelloquin; Guillaume Vial; Nellie Taleux; Christian Slomianny; Ronald J. Wanders; Riekelt H. Houtkooper; Pascale Bellenguer; Ian Max Møller; Eyal Gottlieb; Frederic M. Vaz; Giovanni Manfredi; Patrice X. Petit (1194-1206).
Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III2  + IVn but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.
Keywords: Apoptosis; Barth syndrome; Cardiolipin; Mitochondria; Reactive oxygen species; Respiratory complex;

Metabolic signatures of esophageal cancer: NMR-based metabolomics and UHPLC-based focused metabolomics of blood serum by Xiaoli Zhang; Luan Xu; Jianmin Shen; Bei Cao; Ting Cheng; Tong Zhao; Xiaoyan Liu; Haixia Zhang (1207-1216).
Focused metabolic profiling is a powerful tool for the determination of biomarkers. Here, a more global proton nuclear magnetic resonance (1H NMR)-based metabolomic approach coupled with a relative simple ultra high performance liquid chromatography (UHPLC)-based focused metabolomic approach was developed and compared to characterize the systemic metabolic disturbances underlying esophageal cancer (EC) and identify possible early biomarkers for clinical prognosis. Serum metabolic profiling of patients with EC (n = 25) and healthy controls (n = 25) was performed by using both 1H NMR and UHPLC, and metabolite identification was achieved by multivariate statistical analysis. Using orthogonal projection to least squares discriminant analysis (OPLS-DA), we could distinguish EC patients from healthy controls. The predictive power of the model derived from the UHPLC-based focused metabolomics performed better in both sensitivity and specificity than the results from the NMR-based metabolomics, suggesting that the focused metabolomic technique may be of advantage in the future for the determination of biomarkers. Moreover, focused metabolic profiling is highly simple, accurate and specific, and should prove equally valuable in metabolomic research applications. A total of nineteen significantly altered metabolites were identified as the potential disease associated biomarkers. Significant changes in lipid metabolism, amino acid metabolism, glycolysis, ketogenesis, tricarboxylic acid (TCA) cycle and energy metabolism were observed in EC patients compared with the healthy controls. These results demonstrated that metabolic profiling of serum could be useful as a screening tool for early EC diagnosis and prognosis, and might enhance our understanding of the mechanisms involved in the tumor progression.Display Omitted
Keywords: Metabolomics; Focused metabolomics; Proton nuclear magnetic resonance (1H NMR); Ultra high performance liquid chromatography (UHPLC); Esophageal cancer (EC);

Extracellular chaperones prevent Aβ42-induced toxicity in rat brains by Roberta Cascella; Simona Conti; Francesca Tatini; Elisa Evangelisti; Tania Scartabelli; Fiorella Casamenti; Mark R. Wilson; Fabrizio Chiti; Cristina Cecchi (1217-1226).
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by cognitive decline, formation of the extracellular amyloid β (Aβ42) plaques, neuronal and synapse loss, and activated microglia and astrocytes. Extracellular chaperones, which are known to inhibit amyloid fibril formation and promote clearance of misfolded aggregates, have recently been shown to reduce efficiently the toxicity of HypF-N misfolded oligomers to immortalised cell lines, by binding and clustering them into large species. However, the role of extracellular chaperones on Aβ oligomer toxicity remains unclear, with reports often appearing contradictory. In this study we microinjected into the hippocampus of rat brains Aβ42 oligomers pre-incubated for 1 h with two extracellular chaperones, namely clusterin and α2-macroglobulin. The chaperones were found to prevent Aβ42-induced learning and memory impairments, as assessed by the Morris Water Maze test, and reduce Aβ42-induced glia inflammation and neuronal degeneration in rat brains, as probed by fluorescent immunohistochemical analyses. Moreover, the chaperones were able to prevent Aβ42 colocalisation with PSD-95 at post-synaptic terminals of rat primary neurons, suppressing oligomer cytotoxicity. All such effects were not effective by adding pre-formed oligomers and chaperones without preincubation. Molecular chaperones have therefore the potential to prevent the early symptoms of AD, not just by inhibiting Aβ42 aggregation, as previously demonstrated, but also by suppressing the toxicity of Aβ42 oligomers after they are formed. These findings elect them as novel neuroprotectors against amyloid-induced injury and excellent candidates for the design of therapeutic strategies against AD.
Keywords: Extracellular chaperone; Amyloid neurotoxicity; Hippocampal injury; Learning impairment; Memory injury; Aβ42/PSD-95 colocalisation;

Minocycline, levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease by Anubhuti Dixit; Garima Srivastava; Divya Verma; Manisha Mishra; Pradhyumna Kumar Singh; Om Prakash; Mahendra Pratap Singh (1227-1240).
Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes.
Keywords: Parkinsonism; Mitochondrial proteome; Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin; Levodopa; Minocycline;

Tau hyperphosphorylation and increased BACE1 and RAGE levels in the cortex of PPARβ/δ-null mice by Emma Barroso; Jaume del Valle; David Porquet; Ana M. Vieira Santos; Laia Salvadó; Rosalía Rodríguez-Rodríguez; Patrícia Gutiérrez; Marta Anglada-Huguet; Jordi Alberch; Antoni Camins; Xavier Palomer; Mercè Pallàs; Liliane Michalik; Walter Wahli; Manuel Vázquez-Carrera (1241-1248).
The role of peroxisome proliferator activator receptor (PPAR)β/δ in the pathogenesis of Alzheimer's disease has only recently been explored through the use of PPARβ/δ agonists. Here we evaluated the effects of PPARβ/δ deficiency on the amyloidogenic pathway and tau hyperphosphorylation. PPARβ/δ-null mice showed cognitive impairment in the object recognition task, accompanied by enhanced DNA-binding activity of NF-κB in the cortex and increased expression of IL-6. In addition, two NF-κB-target genes involved in β-amyloid (Aβ) synthesis and deposition, the β site APP cleaving enzyme 1 (Bace1) and the receptor for advanced glycation endproducts (Rage), respectively, increased in PPARβ/δ-null mice compared to wild type animals. The protein levels of glial fibrillary acidic protein (GFAP) increased in the cortex of PPARβ/δ-null mice, which would suggest the presence of astrogliosis. Finally, tau hyperphosphorylation at Ser199 and enhanced levels of PHF-tau were associated with increased levels of the tau kinases CDK5 and phospho-ERK1/2 in the cortex of PPARβ/δ−/− mice.Collectively, our findings indicate that PPARβ/δ deficiency results in cognitive impairment associated with enhanced inflammation, astrogliosis and tau hyperphosphorylation in the cortex.
Keywords: PPARβ/δ; BACE1; RAGE; Tau; ERK1/2; Cortex;

Impairment of proteostasis network in Down syndrome prior to the development of Alzheimer's disease neuropathology: Redox proteomics analysis of human brain by Fabio Di Domenico; Raffaella Coccia; Annalisa Cocciolo; M. Paul Murphy; Giovanna Cenini; Elizabeth Head; D. Allan Butterfield; Alessandra Giorgi; Maria Eugenia Schinina; Cesare Mancuso; Chiara Cini; Marzia Perluigi (1249-1259).
DS is the most frequent genetic cause of intellectual disability characterized by the anomalous presence of three copies of chromosome 21. One of the peculiar features of DS is the onset of Alzheimer's disease neuropathology after the age of 40 years characterized by deposition of senile plaques and neurofibrillary tangles. Growing studies demonstrated that increased oxidative damage, accumulation of unfolded/damaged protein aggregates and dysfunction of intracellular degradative system are key players in neurodegenerative processes. In this study, redox proteomics approach was used to analyze the frontal cortex from DS subjects under the age of 40 compared with age-matched controls, and proteins found to be increasingly carbonylated were identified. Interestingly, our results showed that oxidative damage targets specifically different components of the intracellular quality control system such as GRP78, UCH-L1, V0-ATPase, cathepsin D and GFAP that couples with decreased activity of the proteasome and autophagosome formation observed. We also reported a slight but consistent increase of Aβ 1–42 SDS- and PBS-soluble form and tau phosphorylation in DS versus CTR. We suggest that disturbance in the proteostasis network could contribute to the accumulation of protein aggregates, such as amyloid deposits and NFTs, which occur very early in DS. It is likely that a sub-optimal functioning of degradative systems occur in DS neurons, which in turn provide the basis for further accumulation of toxic protein aggregates. The results of this study suggest that oxidation of protein members of the proteostatis network is an early event in DS and might contribute to neurodegenerative phenomena.
Keywords: Down syndrome; Oxidative stress; Autophagy; Proteasome; Alzheimer disease; Trisomy 21;

Erythropoietin is a hypoxia inducible factor-induced protective molecule in experimental autoimmune neuritis by Bangwei Luo; Man Jiang; Xiaofeng Yang; Zhiyuan Zhang; Jian Xiong; Hermann J. Schluesener; Zhiren Zhang; Yuzhang Wu (1260-1270).
Experimental autoimmune neuritis (EAN), an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system, is characterized by self-limitation. Here we investigated the regulation and contribution of erythropoietin (EPO) in EAN self-limitation. In EAN sciatic nerves, hypoxia, and protein and mRNA levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, EPO and EPO receptor (EPOR) were induced in parallel at disease peak phase but reduced at recovery periods. Further, the deactivation of HIF reduced EAN-induced EPO/EPOR upregulation in EAN, suggesting the central contribution of HIF to EPO/EPOR induction. The deactivation of EPOR signalling exacerbated EAN progression, implying that endogenous EPO contributed to EAN recovery. Exogenous EPO treatment greatly improved EAN recovery. In addition, EPO was shown to promote Schwann cell survival and myelin production. In EAN, EPO treatment inhibited lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3+/CD4+ regulatory T cells and decrease of IFN-γ+/CD4+ Th1 cells. Furthermore, EPO inhibited inflammatory macrophage activation and promoted its phagocytic activity. In summary, our data demonstrated that EPO was induced in EAN by HIF and contributed to EAN recovery, and endogenous and exogenous EPO could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that EPO contributes to the self-recovery of EAN and could be a potent candidate for treatment of autoimmune neuropathies.
Keywords: Erythropoietin; Experimental autoimmune neuritis; Inflammation; Schwann cell; T cell; Macrophage;

Many proteins exhibit propensities to form fibrillar aggregates called amyloids that are rich in β-sheet structures. Abnormal accumulation of amyloids in the brain and spinal cords is well known as a major pathological change in neurodegenerative diseases; therefore, amyloids have long been considered as disease culprits formed via protein misfolding and should be avoided in healthy cells. Recently, however, increasing numbers of proteins have been identified that require formation of fibrillar states for exertion of their physiological functions, and the critical roles of such functional amyloids include a molecular switch for environmental adaptation, a structural template for catalysis, and a regulator of intracellular signaling. Protein amyloids will, therefore, be more prevailed in our physiologies than we have expected so far. Here, we have reviewed recent studies on such regulatory roles of protein fibrillar aggregates in various physiologies and further discussed possible relations of functional to pathological amyloids.
Keywords: Amyloid; Functional amyloid; Protein aggregate; Neurodegenerative disease; RNA granule;

Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia by Thomas J. McCorvie; Tyler J. Gleason; Judith L. Fridovich-Keil; David J. Timson (1279-1293).
Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.Display Omitted
Keywords: GALT; Yeast model; Disease associated mutation; Stability; Substrate binding; Protein misfolding;

miR-135a targets IRS2 and regulates insulin signaling and glucose uptake in the diabetic gastrocnemius skeletal muscle by Priyanka Agarwal; Rohit Srivastava; Arvind K. Srivastava; Shakir Ali; Malabika Datta (1294-1303).
Although aberrant miRNA signatures are associated with diabetes, yet, the status and role of altered miRNAs in the diabetic skeletal muscle is currently poorly understood. Here, we report that 41 miRNAs are altered in the diabetic gastrocnemius skeletal muscle and of these, miR-135a that is identified as a critical regulator of myogenesis, is significantly up-regulated. IRS2 is predicted as its potential putative target and its levels are down-regulated in the diabetic gastrocnemius skeletal muscle. In C2C12 cells, while miR-135a levels decreased during differentiation, IRS2 levels were up-regulated. miR-135a significantly reduced IRS2 protein levels and its 3′UTR luciferase reporter activity and these were blunted by the miR-135a inhibitor and mutation in the miR-135a binding site. Knock-down of endogenous miR-135a levels increased IRS2 at the mRNA and protein levels. miR-135a also attenuated insulin stimulated phosphorylation and activation of PI3Kp85α and Akt and glucose uptake. miR-135a levels were also found to be elevated in the human diabetic skeletal muscle. In-vivo silencing of miR-135a alleviated hyperglycemia, improved glucose tolerance and significantly restored the levels of IRS2 and p-Akt in the gastrocnemius skeletal muscle of db/db mice without any effect on their hepatic levels. These suggest that miR-135a targets IRS2 levels by binding to its 3′UTR and this interaction regulates skeletal muscle insulin signaling.
Keywords: miR-135a; Diabetes; IRS2; Insulin; Microarray; Skeletal muscle;

Mutations in mitochondrial ribosomal protein MRPL12 leads to growth retardation, neurological deterioration and mitochondrial translation deficiency by Valérie Serre; Agata Rozanska; Marine Beinat; Dominique Chretien; Nathalie Boddaert; Arnold Munnich; Agnès Rötig; Zofia M. Chrzanowska-Lightowlers (1304-1312).
Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2–q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent.
Keywords: Mitochondria; Mitoribosome; Protein synthesis; Disease; OXPHOS defect;

Regulation of skeletal muscle oxidative phenotype by classical NF-κB signalling by A.H.V. Remels; H.R. Gosker; J. Bakker; D.C. Guttridge; A.M.W.J. Schols; R.C.J. Langen (1313-1325).
Impairments in skeletal muscle oxidative phenotype (OXPHEN) have been linked to the development of insulin resistance, metabolic inflexibility and progression of the metabolic syndrome and have been associated with progressive disability in diseases associated with chronic systemic inflammation. We previously showed that the inflammatory cytokine tumour necrosis factor-α (TNF-α) directly impairs muscle OXPHEN but underlying molecular mechanisms remained unknown. Interestingly, the inflammatory signalling pathway classical nuclear factor-κB (NF-κB) is activated in muscle in abovementioned disorders. Therefore, we hypothesised that muscle activation of classical NF-κB signalling is sufficient and required for inflammation-induced impairment of muscle OXPHEN.Myotubes from mouse and human muscle cell lines were subjected to activation or blockade of the classical NF-κB pathway. In addition, wild-type and MISR (muscle-specific inhibition of classical NF-κB) mice were injected intra-muscularly with TNF-α. Markers and key regulators of muscle OXPHEN were investigated.Classical NF-κB activation diminished expression of oxidative phosphorylation (OXPHOS) sub-units, slow myosin heavy chain expression, activity of mitochondrial enzymes and potently reduced intra-cellular ATP levels. Accordingly, PGC-1/PPAR/NRF-1/Tfam signalling, the main pathway controlling muscle OXPHEN, was impaired upon classical NF-κB activation which required intact p65 trans-activation domains and depended on de novo gene transcription. Unlike wild-type myotubes, IκBα-SR myotubes (blocked classical NF-κB signalling) were refractory to TNF-α-induced impairments in OXPHEN and its regulation by the PGC-1/PPAR/NRF-1/Tfam cascade. In line with in vitro data, NF-κB blockade in vivo abrogated TNF-α-induced reductions in PGC-1α expression.Classical NF-κB activation impairs skeletal muscle OXPHEN.
Keywords: Skeletal muscle; Inflammation; Mitochondria; Oxidative metabolism; Classical NF-κB;

Iron uptake in quiescent and inflammation-activated astrocytes: A potentially neuroprotective control of iron burden by Ilaria Pelizzoni; Daniele Zacchetti; Alessandro Campanella; Fabio Grohovaz; Franca Codazzi (1326-1333).
Astrocytes play a crucial role in proper iron handling within the central nervous system. This competence can be fundamental, particularly during neuroinflammation, and neurodegenerative processes, where an increase in iron content can favor oxidative stress, thereby worsening disease progression. Under these pathological conditions, astrocytes undergo a process of activation that confers them either a beneficial or a detrimental role on neuronal survival. Our work investigates the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes. Our data confirm that the main source of iron is the non-transferrin-bound iron (NTBI) and show the involvement of two different routes for its entry: the resident transient receptor potential (TRP) channels in quiescent astrocytes and the de novo expressed divalent metal transporter 1 (DMT1) in activated astrocytes, which accounts for a potentiation of iron entry. Overall, our data suggest that at rest, but even more after activation, astrocytes have the potential to buffer the excess of iron, thereby protecting neurons from iron overload. These findings further extend our understanding of the protective role of astrocytes under the conditions of iron-mediated oxidative stress observed in several neurodegenerative conditions.
Keywords: Astrocytes; Non-transferrin-bound iron uptake; DMT1; Activation process; TRP channels; Neuroinflammation;

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death by Valentina Parra; Francisco Moraga; Jovan Kuzmicic; Camila López-Crisosto; Rodrigo Troncoso; Natalia Torrealba; Alfredo Criollo; Jessica Díaz-Elizondo; Beverly A. Rothermel; Andrew F.G. Quest; Sergio Lavandero (1334-1344).
Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2 + overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2 + levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2 + influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2 + buffer capacity. These biochemical events increase cytosolic Ca2 + levels and trigger cardiomyocyte death via the activation of calpains.Display Omitted
Keywords: Ceramide; Metabolism; Mitochondrial dynamics; Ca2 +; Cell death; Cardiomyocyte;

Lysine deacetylases and mitochondrial dynamics in neurodegeneration by Pedro Guedes-Dias; Jorge M.A. Oliveira (1345-1359).
Lysine acetylation is a key post-translational modification known to regulate gene transcription, signal transduction, cellular transport and metabolism. Lysine deacetylases (KDACs), including classical KDACs (a.k.a. histone deacetylases; HDACs) and sirtuins (SIRTs), are emerging therapeutic targets in neurodegeneration. Given the strong link between abnormal mitochondrial dynamics and neurodegenerative disorders (e.g. in Alzheimer, Parkinson and Huntington diseases), here we examine the evidence for KDAC-mediated regulation of mitochondrial biogenesis, fission–fusion, movement and mitophagy. Mitochondrial biogenesis regulation was reported for SIRT1, SIRT3, and class IIa KDACs, mainly via PGC-1alpha modulation. SIRT1 or SIRT3 overexpression rescued mitochondrial density and fission–fusion balance in neurodegeneration models. Mitochondrial fission decreased with pan-classical-KDAC inhibitors and increased with nicotinamide (pan-sirtuin-inhibitor/activator depending on concentration and NAD+ conversion). Mitochondrial movement increased with HDAC6 inhibition, but this is not yet reported for the other tubulin deacetylase SIRT2. Inhibition of HDAC6 or SIRT2 was reported neuroprotective. Mitophagy is assisted by the HDAC6 ubiquitin-binding and autophagosome–lysosome fusion promoting activities, and was also associated with SIRT1 activation. In summary, KDACs can potentially modulate multiple components of mitochondrial dynamics, however, several key points require clarification. The SIRT1-biogenesis connection relies heavily in controversial caloric restriction (CR) regimes or CR-mimetic drugs, and appears cell-type dependent, recommending caution before linking SIRT1 activation with general neuroprotection. Future studies should clarify mitochondrial fission–fusion regulation by KDACs, and the interplay between HDAC6 and SIRT1 in mitophagy. Also, further studies are required to ascertain whether HDAC6 inhibition to enhance mitochondrial trafficking does not compromise autophagy or clearance of misfolded proteins in neurodegenerative disorders.
Keywords: Mitochondria; HDAC; Sirtuin; Mitochondrial dynamics; Biogenesis; Mitophagy;