BBA - Molecular Basis of Disease (v.1852, #10PA)

Loss of PHLPP protects against colitis by inhibiting intestinal epithelial cell apoptosis by Yang-An Wen; Xin Li; Tatiana Goretsky; Heidi L. Weiss; Terrence A. Barrett; Tianyan Gao (2013-2023).
A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis.
Keywords: PHLPP; Knockout mouse; Intestine epithelial cell apoptosis; Akt; Inflammatory bowel disease;

Post-transcriptional regulation of cardiac sodium channel gene SCN5A expression and function by miR-192-5p by Yuanyuan Zhao; Yuan Huang; Weihua Li; Zhijie Wang; Shaopeng Zhan; Mengchen Zhou; Yufeng Yao; Zhipeng Zeng; Yuxi Hou; Qiuyun Chen; Xin Tu; Qing K. Wang; Zhengrong Huang (2024-2034).
The SCN5A gene encodes cardiac sodium channel Nav1.5 and causes lethal ventricular arrhythmias/sudden death and atrial fibrillation (AF) when mutated. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and involved in the pathogenesis of many diseases. However, little is known about the regulation of SCN5A by miRNAs. Here we reveal a novel post-transcriptional regulatory mechanism for expression and function of SCN5A/Nav1.5 via miR-192-5p. Bioinformatic analysis revealed that the 3′-UTR of human and rhesus SCN5A, but not elephant, pig, rabbit, mouse, and rat SCN5A, contained a target binding site for miR-192-5p and dual luciferase reporter assays showed that the site was critical for down-regulation of human SCN5A. With Western blot assays and electrophysiological studies, we demonstrated that miR-192-5p significantly reduced expression of SCN5A and Nav1.5 as well as peak sodium current density INa generated by Nav1.5. Notably, in situ hybridization, immunohistochemistry and real-time qPCR analyses showed that miR-192-5p was up-regulated in tissue samples from AF patients, which was associated with down-regulation of SCN5A/Nav1.5. These results demonstrate an important post-transcriptional role of miR-192-5p in post-transcriptional regulation of Nav1.5, reveal a novel role of miR-192-5p in cardiac physiology and disease, and provide a new target for novel miRNA-based antiarrhythmic therapy for diseases with reduced INa .
Keywords: Atrial fibrillation; Cardiac sodium channel Nav1.5; SCN5A; MicroRNA; MiR-192-5p;

GPER: A new tool to protect dopaminergic neurons? by Agustina Bessa; Filipa Lopes Campos; Rita Alexandra Videira; Julieta Mendes-Oliveira; Diogo Bessa-Neto; Graça Baltazar (2035-2041).
Parkinson's disease (PD) is characterized by a selective degeneration of nigrostriatal dopaminergic pathway. Epidemiological studies revealed a male predominance of the disease that has been attributed to the female steroid hormones, mainly the estrogen. Estrogen neuroprotective effects have been shown in several studies, however the mechanisms responsible by these effects are still unclear. Previous data from our group revealed that glial cell line-derived neurotrophic factor (GDNF) is crucial to the dopaminergic protection provided by 17β-estradiol, and also suggest that the intracellular estrogen receptors (ERs) are not required for that neuroprotective effects.The present study aimed to investigate the contribution of the G protein-coupled ER (GPER) activation in estrogen-mediated dopaminergic neuroprotection against an insult induced by 1-methyl-4-phenylpyridinium (MPP+), and whether GPER neuroprotective effects involve the regulation of GDNF expression. Using primary mesencephalic cultures, we found that GPER activation protects dopaminergic neurons from MPP+ toxicity in an extent similar to the promoted by a 17β-estradiol. Moreover, GPER activation promotes an increase in GDNF levels. Both, GDNF antibody neutralization or RNA interference-mediated GDNF knockdown prevented the GPER-mediated dopaminergic protection verified in mesencephalic cultures challenged with MPP+. Overall, these results revealed that G1, a selective agonist of GPER, is able to protect dopaminergic neurons and that GDNF overexpression is a key feature to GPER induced the neuroprotective effects.
Keywords: Parkinson's disease; GPER; GDNF; Dopaminergic neurons; Estradiol;

Functional analysis of SERCA1b, a highly expressed SERCA1 variant in myotonic dystrophy type 1 muscle by Yimeng Zhao; Haruo Ogawa; Shin-Ichiro Yonekura; Hiroaki Mitsuhashi; Satomi Mitsuhashi; Ichizo Nishino; Chikashi Toyoshima; Shoichi Ishiura (2042-2047).
Myotonic dystrophy type 1 (DM1) is a genetic disorder in which multiple genes are aberrantly spliced. Sarco/endoplasmic reticulum Ca2 +-ATPase 1 (SERCA1) is one of these genes, and it encodes a P-type ATPase. SERCA1 transports Ca2 + from the cytosol to the lumen, and is involved in muscular relaxation. It has two splice variants (SERCA1a and SERCA1b) that differ in the last eight amino acids, and the contribution of these variants to DM1 pathology is unclear. Here, we show that SERCA1b protein is highly expressed in DM1 muscle tissue, mainly localised at fast twitch fibres. Additionally, when SERCA1a and SERCA1b were overexpressed in cells, we found that the ATPase and Ca2 + uptake activity of SERCA1a was almost double that of SERCA1b. Although the affinity for both ATP and Ca2 + was similar between the two variants, SERCA1b was more sensitive to the inner microsomal environment. Thus, we hypothesise that aberrant expression of SERCA1b in DM1 patients is the cause of abnormal intracellular Ca2 + homeostasis.
Keywords: SERCA1a; SERCA1b; Myotonic dystrophy; P-type ATPase; Alternative splicing;

Both endothelin-1 (ET-1) and cAMP are implicated for inducing insulin resistance. Since we have shown previously that there is a crosstalk between ET-1 and cAMP signaling pathways in regulating glucose uptake in 3T3-L1 adipocytes, we extended our investigation in this study on whether they may have a synergistic effect on inducing insulin resistance. Our results showed that it was indeed the case. Insulin-stimulated glucose uptake, phosphorylation of PKB, IRS-1-associated PI3K, and IRS-1 tyrosine phosphorylation were all inhibited by ET-1 and 8-bromo cAMP in a synergistic manner. IRS-1 protein levels were similarly decreased by ET-1 and 8-bromo cAMP, attributable to suppressed mRNA expression. In addition, after correction for the loss in IRS-1 protein, the inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation or IRS-1-associated PI3K was mainly caused by cAMP. Moreover, whereas IRS-2 protein levels were increased by cAMP or ET-1 plus cAMP, insulin-stimulated IRS-2-associated PI3K activities were abolished by both treatments. Furthermore, ET-1 and β-adrenergic agonists had similar synergistic inhibition on insulin-stimulated glucose uptake. In conclusion, we have shown that ET-1 and cAMP may synergistically induce insulin resistance in adipocytes via inhibiting IRS-1 expression as well as insulin-stimulated IRS-1/IRS-2 activities.
Keywords: Endothelin-1; cAMP; Insulin resistance; Insulin receptor substrate-1; Insulin receptor substrate-2; Adipocytes; β-Adrenergic agonists;

Adaptive changes in amino acid metabolism permit normal longevity in mice consuming a low-carbohydrate ketogenic diet by Nicholas Douris; Tamar Melman; Jordan M. Pecherer; Pavlos Pissios; Jeffrey S. Flier; Lewis C. Cantley; Jason W. Locasale; Eleftheria Maratos-Flier (2056-2065).
Ingestion of very low-carbohydrate ketogenic diets (KD) is associated with weight loss, lowering of glucose and insulin levels and improved systemic insulin sensitivity. However, the beneficial effects of long-term feeding have been the subject of debate. We therefore studied the effects of lifelong consumption of this diet in mice. Complete metabolic analyses were performed after 8 and 80 weeks on the diet. In addition we performed a serum metabolomic analysis and examined hepatic gene expression. Lifelong consumption of KD had no effect on morbidity or mortality (KD vs. Chow, 676 vs. 630 days) despite hepatic steatosis and inflammation in KD mice. The KD fed mice lost weight initially as previously reported (Kennnedy et al., 2007) and remained lighter and had less fat mass; KD consuming mice had higher levels of energy expenditure, improved glucose homeostasis and higher circulating levels of β-hydroxybutyrate and triglycerides than chow-fed controls. Hepatic expression of the critical metabolic regulators including fibroblast growth factor 21 were also higher in KD-fed mice while expression levels of lipogenic enzymes such as stearoyl-CoA desaturase-1 was reduced. Metabolomic analysis revealed compensatory changes in amino acid metabolism, primarily involving down-regulation of catabolic processes, demonstrating that mice eating KD can shift amino acid metabolism to conserve amino acid levels. Long-term KD feeding caused profound and persistent metabolic changes, the majority of which are seen as health promoting, and had no adverse effects on survival in mice.
Keywords: Ketogenic diet; Liver; Ketogenesis; Hepatic steatosis; Free fatty acid metabolism; Metabolomics; Mass spectrometry;

Impaired enzymatic defensive activity, mitochondrial dysfunction and proteasome activation are involved in RTT cell oxidative damage by Carlo Cervellati; Claudia Sticozzi; Arianna Romani; Giuseppe Belmonte; Domenico De Rasmo; Anna Signorile; Franco Cervellati; Chiara Milanese; Pier Giorgio Mastroberardino; Alessandra Pecorelli; Vinno Savelli; Henry J. Forman; Joussef Hayek; Giuseppe Valacchi (2066-2074).
A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment.
Keywords: Oxidative stress; NADPH oxidase; Glutathione peroxidase; Superoxide dismutase thioredoxin-reductases; 4HNE;

Cardiomyocyte–fibroblast interaction contributes to diabetic cardiomyopathy in mice: Role of HMGB1/TLR4/IL-33 axis by Aibin Tao; Jia Song; Ting Lan; Xuemei Xu; Peter Kvietys; Raymond Kao; Claudio Martin; Tao Rui (2075-2085).
Diabetic cardiomyopathy (DiCM) is characterized by myocardial fibrosis and dysfunction. In rodent models of diabetes myocardial HMGB1 increases while IL-33 decreases. The major cardiac cell type expressing HMGB1 is the myocyte while the primary IL-33 expressing cell is the fibroblast. The aim of this study was to delineate the extracellular communication pathway(s) between cardiomyocytes and fibroblasts that contributes to murine DiCM. The streptozotocin (STZ)-induced murine model of diabetes and a cardiomyocyte/fibroblast co-culture challenged with high glucose were used. In STZ mice, myocardial HMGB1 expression was increased while IL-33 expression decreased (immunofluorescence and Western blot). In addition, STZ mice had an increased myocardial collagen deposition and myocardial dysfunction (pressure-volume loop analysis). An HMGB1 inhibitor (A-box) or exogenous IL-33 prevented the myocardial collagen deposition and dysfunction. In the cardiomyocyte/fibroblast co-culture model, HG increased cardiomyocyte HMGB1 secretion, decreased fibroblast IL-33 expression, and increased fibroblast collagen I production. Further, using A-box and HMGB1 shRNA transfected myocytes, we found that cardiomyocyte-derived HMGB1 dramatically potentiated the HG-induced down-regulation of IL-33 and the increase in collagen I expression in the fibroblasts. The potentiating effects of the cardiomyocytes was diminished when toll-like receptor 4 deficient (TLR4−/−) fibroblasts were co-cultured with wild-type myocytes. Finally, TLR4−/− mice with diabetes had increased myocardial expression of HMGB1, but failed to down-regulate IL-33. The diabetes-induced myocardial collagen deposition and cardiac dysfunction were significantly attenuated in TLR4−/− mice. In conclusion, our findings support a role for “cardiomyocyte HMGB1–fibroblast TLR4/IL-33 axis” in the development of myocardial fibrosis and dysfunction in a murine model of diabetes.
Keywords: Myocardial fibrosis; Diabetes mellitus; HMGB1; IL-33; TLR4;

Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (ENT3) have been identified in patients with pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID). ENT3 is a member of the equilibrative nucleoside transporter (ENT) family whose primary function is mediating transport of nucleosides and nucleobases. The aims of this study were to characterise ENT3 expression in islet β-cells and identify the effects of its depletion on β-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 β-cells. Immunohistochemistry using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of β-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 β-cells to MitoTracker and an ENT3 antibody showed co-localisation of ENT3 to β-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in β-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in β-cells increased mitochondrial DNA content and promoted an energy crisis characterised by enhanced ATP-linked respiration and proton leak. Finally, inhibition of ENT3 activity by dypridamole and depletion of ENT3 by siRNA-induced knockdown resulted in increased caspase 3/7 activities in β-cells. These observations demonstrate that ENT3 is predominantly expressed by islet β-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced β-cell apoptosis. Thus, apoptotic loss of islet β-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional ENT3 mutations.
Keywords: Islets of Langerhans; β-Cells; Equilibrative nucleoside transporter 3; Diabetes; Mitochondria; Apoptosis;

ER-to-mitochondria miscommunication and metabolic diseases by Camila López-Crisosto; Roberto Bravo-Sagua; Marcelo Rodriguez-Peña; Claudia Mera; Pablo F. Castro; Andrew F.G. Quest; Beverly A. Rothermel; Mariana Cifuentes; Sergio Lavandero (2096-2105).
Eukaryotic cells contain a variety of subcellular organelles, each of which performs unique tasks. Thus follows that in order to coordinate these different intracellular functions, a highly dynamic system of communication must exist between the various compartments. Direct endoplasmic reticulum (ER)–mitochondria communication is facilitated by the physical interaction of their membranes in dedicated structural domains known as mitochondria-associated membranes (MAMs), which facilitate calcium (Ca2 +) and lipid transfer between organelles and also act as platforms for signaling. Numerous studies have demonstrated the importance of MAM in ensuring correct function of both organelles, and recently MAMs have been implicated in the genesis of various human diseases. Here, we review the salient structural features of interorganellar communication via MAM and discuss the most common experimental techniques employed to assess functionality of these domains. Finally, we will highlight the contribution of MAM to a variety of cellular functions and consider the potential role of MAM in the genesis of metabolic diseases. In doing so, the importance for cell functions of maintaining appropriate communication between ER and mitochondria will be emphasized.Display Omitted
Keywords: Interorganelle communication; Endoplasmic reticulum; Mitochondria; Mitochondria-associated membranes; Mitochondrial metabolism; Metabolic diseases;

Lack of LCAT reduces the LPS-neutralizing capacity of HDL and enhances LPS-induced inflammation in mice by Peristera-Ioanna Petropoulou; Jimmy F.P. Berbée; Vassilios Theodoropoulos; Aikaterini Hatziri; Panagiota Stamou; Eleni A. Karavia; Alexandros Spyridonidis; Iordanes Karagiannides; Kyriakos E. Kypreos (2106-2115).
HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. As lecithin–cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat −/−) mice present an increased LPS-induced inflammatory response. LPS (100 μg/kg body weight)-induced cytokine response in Lcat −/− mice was markedly enhanced and prolonged compared to wild-type mice. Importantly, reintroducing LCAT expression using adenovirus-mediated gene transfer reverted their phenotype to that of wild-type mice. Ex vivo stimulation of whole blood with LPS (1–100 ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons, VLDL or the lipid-free protein fraction of Lcat −/− mice, had a reduced capacity to attenuate the LPS-induced TNFα response. Analysis of apolipoprotein composition revealed that LCAT-deficient HDL lacks significant amounts of ApoA-I and ApoA-II and is primarily composed of ApoE, while HDL from Apoa1 −/− mice is highly enriched in ApoE and ApoA-II. ApoA-I-deficiency did not affect the capacity of HDL to neutralize LPS, though Apoa1 −/− mice showed a pronounced LPS-induced cytokine response. Additional immunophenotyping showed that Lcat −/− , but not Apoa1 −/− mice, have markedly increased circulating monocyte numbers as a result of increased Cd11b+Ly6Cmed monocytes, whereas ‘pro-inflammatory’ Cd11b+Ly6Chi monocytes were reduced. In line with this observation, peritoneal macrophages of Lcat −/− mice showed a markedly dampened LPS-induced TNFα response. We conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL and increased monocyte number.
Keywords: Lecithin–cholesterol acyltransferase; Apolipoprotein A-I; High-density lipoprotein; LPS; Inflammation; Mice;

Decrease in APP and CP mRNA expression supports impairment of iron export in Alzheimer's disease patients by Cláudia Guerreiro; Bruno Silva; Ângela C. Crespo; Liliana Marques; Sónia Costa; Ângela Timóteo; Erica Marcelino; Carolina Maruta; Arminda Vilares; Mafalda Matos; Frederico Simões Couto; Paula Faustino; Ana Verdelho; Manuela Guerreiro; Ana Herrero; Cristina Costa; Alexandre de Mendonça; Madalena Martins; Luciana Costa (2116-2122).
Alzheimer's disease (AD) is a neurodegenerative disorder of still unknown etiology and the leading cause of dementia worldwide. Besides its main neuropathological hallmarks, a dysfunctional homeostasis of transition metals has been reported to play a pivotal role in the pathogenesis of this disease. Dysregulation of iron (Fe) metabolism in AD has been suggested, particularly at the level of cellular iron efflux. Herein, we intended to further clarify the molecular mechanisms underlying Fe homeostasis in AD. In order to achieve this goal, the expression of specific Fe metabolism-related genes directly involved in Fe regulation and export was assessed in peripheral blood mononuclear cells (PBMCs) from 73 AD patients and 74 controls by quantitative PCR. The results obtained showed a significant decrease in the expression of aconitase 1 (ACO1; P  = 0.007); ceruloplasmin (CP; P  < 0.001) and amyloid-beta precursor protein (APP; P  = 0.006) genes in AD patients compared with healthy volunteers. These observations point out to a significant downregulation in the expression of genes associated with ferroportin-mediated cellular Fe export in PBMCs from AD patients, when compared to controls. Taken together, these findings support previous studies suggesting impairment of Fe homeostasis in AD, which may lead to cellular Fe retention and oxidative stress, a typical feature of this disease.
Keywords: Alzheimer's disease; Iron homeostasis; Cellular iron export; Gene expression;

Inhibition of protein kinase CK2 suppresses tumor necrosis factor (TNF)-α-induced leukocyte–endothelial cell interaction by Emmanuel Ampofo; Jeannette Rudzitis-Auth; Indra N. Dahmke; Oliver G. Rössler; Gerald Thiel; Mathias Montenarh; Michael D. Menger; Matthias W. Laschke (2123-2136).
Inflammatory endothelial processes are regulated by the nuclear factor-κB (NF-κB) pathway, which involves phosphorylation of p65. Because p65 is a substrate of CK2, we herein investigated, whether this pleiotropic protein kinase may be a beneficial anti-inflammatory target. For this purpose, we analyzed in human dermal microvascular endothelial cells (HDMEC) the effect of CK2 inhibition by quinalizarin and CX-4945 on cell viability, adhesion molecule expression and NF-κB pathway activation. Leukocyte binding to HDMEC was assessed in an in vitro adhesion assay. Dorsal skinfold chambers in BALB/c mice were used to study leukocyte–endothelial cell interaction and leukocyte transmigration by means of repetitive intravital fluorescence microscopy and immunohistochemistry. We found that quinalizarin and CX-4945 effectively suppressed the activity of CK2 in HDMEC without affecting their viability. This was associated with a significant down-regulation of tumor necrosis factor (TNF)-α-induced E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression due to a reduction of shuttling, phosphorylation and transcriptional activity of the NF-κB complex. In consequence, leukocyte binding to quinalizarin- and CX-4945-treated HDMEC was diminished. Finally, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in dorsal skinfold chambers exposed to TNF-α in vivo. These findings indicate that CK2 is a key regulator of leukocyte–endothelial cell interaction in inflammation by regulating the expression of E-selectin, ICAM-1 and VCAM-1 via affecting the transcriptional activity of the NF-κB complex. Accordingly, CK2 represents a promising target for the development of novel anti-inflammatory drugs.
Keywords: Leukocyte adhesion; NF-κB; ICAM-1; VCAM-1; Dorsal skinfold chamber; Intravital fluorescence microscopy;

Suppression of RANKL-induced osteoclast differentiation by cilostazol via SIRT1-induced RANK inhibition by So Youn Park; Sung Won Lee; Hye Young Kim; Sang Yeob Lee; Won Suk Lee; Ki Whan Hong; Chi Dae Kim (2137-2144).
Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage hematopoietic lineages and degrade bone matrix by secretion of lytic enzymes. The regulation of osteoclast differentiation provides a potential strategy for treatment of bone-lytic damage. In this study, cilostazol, an inhibitor of type III phosphodiesterase, inhibited RANKL [receptor activator of nuclear factor kappa B (RANK) ligand]-induced RANK expression in bone marrow-derived monocyte/macrophage precursors (BMMs) and Raw 264.7 cells by inhibiting PU.1 via SIRT1 activation. RANKL-induced RANK expression was attenuated by cilostazol and rSIRT1 in Raw 264.7 cells, and these were blocked by sirtinol. In line with these, cilostazol elevated SIRT1 mRNA and protein levels in 12–24 h and increased SIRT1 activity, and these effects were inhibited by sirtinol. Furthermore, the RANKL-induced nuclear expression of PU.1, a transcription factor required for macrophage differentiation, was suppressed by cilostazol. Additionally, marked RANKL-induced RANK immunofluorescence staining in Raw 264.7 cells was attenuated by cilostazol and rSIRT1, and both attenuations were prevented by sirtinol. Extensive RANK staining of knee synovial tissues in a mouse model of collagen-induced arthritis (CIA) was markedly reduced by cilostazol (30 mg/kg/day). In line with these results, both RANKL- and M-CSF-induced differentiation of BMMs to multinucleated TRAP+ giant cells and resorption pit formation were inhibited by cilostazol associated with a decrease in TRAP (a marker enzyme of osteoclasts) activity. In conclusion, cilostazol activates SIRT1, which suppresses the nuclear translocation of PU.1, and thus, inhibits RANKL-stimulated RANK expression and causes anti-osteoclast formation in BMMs in vitro and in their murine model of CIA.
Keywords: Rheumatoid arthritis; Osteoclast; RANK; SIRT1; PU.1; Cilostazol;

Cdk5-mediated mitochondrial fission: A key player in dopaminergic toxicity in Huntington's disease by Marta Cherubini; Mar Puigdellívol; Jordi Alberch; Silvia Ginés (2145-2160).
The molecular mechanisms underlying striatal vulnerability in Huntington's disease (HD) are still unknown. However, growing evidence suggest that mitochondrial dysfunction could play a major role. In searching for a potential link between striatal neurodegeneration and mitochondrial defects we focused on cyclin-dependent kinase 5 (Cdk5). Here, we demonstrate that increased mitochondrial fission in mutant huntingtin striatal cells can be a consequence of Cdk5-mediated alterations in Drp1 subcellular distribution and activity since pharmacological or genetic inhibition of Cdk5 normalizes Drp1 function ameliorating mitochondrial fragmentation. Interestingly, mitochondrial defects in mutant huntingtin striatal cells can be worsened by D1 receptor activation a process also mediated by Cdk5 as down-regulation of Cdk5 activity abrogates the increase in mitochondrial fission, the translocation of Drp1 to the mitochondria and the raise of Drp1 activity induced by dopaminergic stimulation. In sum, we have demonstrated a new role for Cdk5 in HD pathology by mediating dopaminergic neurotoxicity through modulation of Drp1-induced mitochondrial fragmentation, which underscores the relevance for pharmacologic interference of Cdk5 signaling to prevent or ameliorate striatal neurodegeneration in HD.
Keywords: Cdk5; Dopaminergic activation; Drp1; Huntingtin; Mitochondrial dynamics;

The deletion of the estrogen receptor α gene reduces susceptibility to estrogen-induced cholesterol cholelithiasis in female mice by Ornella de Bari; Helen H. Wang; Piero Portincasa; Min Liu; David Q.-H. Wang (2161-2169).
Compelling evidence has demonstrated that estrogen is a critical risk factor for gallstone formation and enhances cholesterol cholelithogenesis through the hepatic estrogen receptor α (ERα), but not ERβ. To study the lithogenic mechanisms of estrogen through ERα, we investigated whether the deletion of Erα protects against gallstone formation in ovariectomized (OVX) female mice fed a lithogenic diet and treated with 17β-estradiol (E2) at 0 or 6 μg/day for 56 days. Our results showed that the prevalence of gallstones was reduced from 100% in OVX ERα (+/+) mice to 30% in OVX ERα (−/−) mice in response to high doses of E2 and the lithogenic diet for 56 days. Hepatic cholesterol secretion was significantly diminished in OVX ERα (−/−) mice compared to OVX ERα (+/+) mice even fed the lithogenic diet and treated with E2 for 56 days. These alterations decreased bile lithogenicity by reducing cholesterol saturation index of gallbladder bile. Immunohistochemical studies revealed that ERα was expressed mainly in the gallbladder smooth muscle cells. High levels of E2 impaired gallbladder emptying function mostly through the ERα and cholecystokinin-1 receptor pathway, leading to gallbladder stasis in OVX ERα (+/+) mice. By contrast, gallbladder emptying function was greatly improved in OVX ERα (−/−) mice. This markedly retarded cholesterol crystallization and the growth and agglomeration of solid cholesterol crystals into microlithiasis and stones. In conclusion, the deletion of Erα reduces susceptibility to the formation of E2-induced gallstones by diminishing hepatic cholesterol secretion, desaturating gallbladder bile, and improving gallbladder contraction function in female mice.
Keywords: Bile flow; Bile salts; Biliary secretion; Cholesterol crystallization; Gallbladder motility; Lith gene;

Differential roles of MMP-9 in early and late stages of dystrophic muscles in a mouse model of Duchenne muscular dystrophy by Naoko Shiba; Daigo Miyazaki; Takahiro Yoshizawa; Kazuhiro Fukushima; Yuji Shiba; Yuji Inaba; Michihiro Imamura; Shin'ichi Takeda; Kenichi Koike; Akinori Nakamura (2170-2182).
Matrix metalloprotease (MMP)-9 is an endopeptidase associated with the pathogenesis of Duchenne muscular dystrophy (DMD). The precise function of MMP-9 in DMD has not been elucidated to date. We investigated the effect of genetic ablation of MMP-9 in the mdx mouse model (mdx/Mmp9−/− ). At the early disease stage, the muscles of mdx/Mmp9−/− mice showed reduced necrosis and neutrophil invasion, accompanied by down-regulation of chemokine MIP-2. In addition, muscle regeneration was enhanced, which coincided with increased macrophage infiltration and upregulation of MCP-1, and resulted in increased muscle strength. The mdx/Mmp9−/− mice also displayed accelerated upregulation of osteopontin expression in skeletal muscle at the acute onset phase of dystrophy. However, at a later disease stage, the mice exhibited muscle growth impairment through altered expression of myogenic factors, and increased fibroadipose tissue. These results showed that MMP-9 might have multiple functions during disease progression. Therapy targeting MMP-9 may improve muscle pathology and function at the early disease stage, but continuous inhibition of this protein may result in the accumulation of fibroadipose tissues and reduced muscle strength at the late disease stage.
Keywords: Dystrophin; Fibrosis; MMP-9; MIP-2; MCP-1; Osteopontin;

Sirtuins: double players in Huntington's disease by Luana Naia; A. Cristina Rego (2183-2194).
Sirtuins are a conserved family of NAD+-dependent class III lysine deacetylases, known to regulate longevity. In mammals, the sirtuin family has seven members (SIRT1–7), which vary in enzymatic activity, subcellular distribution and targets. Pharmacological and genetic modulation of SIRTs has been widely spread as a promising approach to slow aging and neurodegenerative processes. Huntington's disease (HD) is a neurodegenerative disorder linked to expression of polyglutamine-expanded huntingtin (HTT) protein for which there is still no disease-reversing treatment. Studies in different animal models provide convincing evidence that SIRT1 protects both cellular and animal models from mutant HTT toxicity, however controversial results were recently reported. Indeed, as a consequence of a variety of SIRT-activation pathways, either activation or inhibition of a specific SIRT appears to be neuroprotective. Therefore, this review summarizes the recent progress and knowledge in sirtuins (particularly SIRT1–3) and their implications for HD treatment.
Keywords: Sirtuins; Lysine deacetylases; Deacetylation; Huntington's disease; Therapeutic targets; Neuroprotection;

Heme oxygenase (HO)-1 confers transient resistance against oxidative damage, including renal ischemia-reperfusion injury (IRI). We investigated the potential protective effect of HO-1 induction in a mouse model of renal IRI induced by bilateral clamping of the kidney arteries. The mice were randomly assigned to five groups to receive an intraperitoneal injection of PBS, hemin (an HO-1 inducer, 100 μmol/kg), hemin + ZnPP (an HO-1 inhibitor, 5 mg/kg), hemin + PD98059 (a MEK-ERK inhibitor, 10 mg/kg) or a sham operation. All of the groups except for the sham-operated group underwent 25 min of ischemia and 24 to 72 h of reperfusion. Renal function and tubular damage were assessed in the mice that received hemin or the vehicle treatment prior to IRI. The renal injury score and HO-1 protein levels were evaluated via H&E and immunohistochemistry staining. Hemin-preconditioned mice exhibited preserved renal cell function (BUN: 40 ± 2 mg/dl, creatinine: 0.53 ± 0.06 mg/dl), and the tubular injury score at 72 h (1.65 ± 0.12) indicated that tubular damage was prevented. Induction of HO-1 induced the phosphorylation of extracellular signal-regulated kinases (ERK) 1/2. However, these effects were abolished with ZnPP treatment. Kidney function (BUN: 176 ± 49 mg/dl, creatinine: 1.54 ± 0.39 mg/dl) increased, and the tubular injury score (3.73 ± 0.09) indicated that tubular damage also increased with ZnPP treatment. HO-1-induced tubular epithelial proliferation was attenuated by PD98059. Our findings suggest that HO-1 preconditioning promotes ERK1/2 phosphorylation and enhances tubular recovery, which subsequently prevents further renal injury.Display Omitted
Keywords: ERK; Hemin; HO-1; Ischemia-reperfusion injury; Tubular epithelial proliferation; Tubular recovery;

High inorganic phosphate concentration inhibits osteoclastogenesis by modulating miR-223 by Eléonore M'Baya-Moutoula; Loïc Louvet; Valérie Metzinger-Le Meuth; Ziad A. Massy; Laurent Metzinger (2202-2212).
Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a common complication of CKD, and uremic toxins have been shown to be instrumental in this process. We have previously shown that miR-223 is increased in smooth muscle cells subjected to the uremic toxin inorganic phosphate (Pi). In the present study we investigated the influence of this miRNA in osteoclastogenesis in order to elucidate its role in the course of CKD-MBD. RT-qPCR demonstrated that high Pi concentration decreased miR-223 expression in differentiated RAW 264.7 cells. Up- and down-regulation of miR-223 was performed using specific pre-miR and anti-miR-223. Differentiation of monocyte/macrophage precursors was assessed by using RAW 264.7 cells and peripheral blood mononuclear cells (PBMC). TRAP activity and bone resorption were used to measure osteoclast activity. Pi induced a marked decrease in osteoclastogenesis in RAW cells and miR-223 levels were concomitantly decreased. Anti-miR-223 treatment inhibited osteoclastogenesis in the same way as Pi. In contrast, overexpression of miR-223 triggered differentiation, as reflected by TRAP activity. We showed that miR-223 affected the expression of its target genes NFIA and RhoB, but also osteoclast marker genes and the Akt signalling pathway, which induces osteoclastogenesis. These results were confirmed by measuring bone resorption activity of human PBMC differentiated into osteoclasts. We thus demonstrate a role of miR-223 in osteoclast differentiation, with rational grounds to use deregulation of this miRNA to selectively increase osteoclast-like activity in calcified vessels of CKD-MBD. This approach could alleviate vascular calcification without altering bone structure.
Keywords: Osteoclast; Vascular; Calcification; MicroRNA (miRNA); Cell differentiation; miR-223;

Age-related changes in the proteostasis network in the brain of the naked mole-rat: Implications promoting healthy longevity by Judy C. Triplett; Antonella Tramutola; Aaron Swomley; Jessime Kirk; Kelly Grimes; Kaitilyn Lewis; Miranda Orr; Karl Rodriguez; Jian Cai; Jon B. Klein; Marzia Perluigi; Rochelle Buffenstein; D. Allan Butterfield (2213-2224).
The naked mole-rat (NMR) is the longest-lived rodent and possesses several exceptional traits: marked cancer resistance, negligible senescence, prolonged genomic integrity, pronounced proteostasis, and a sustained health span. The underlying molecular mechanisms that contribute to these extraordinary attributes are currently under investigation to gain insights that may conceivably promote and extend human health span and lifespan. The ubiquitin–proteasome and autophagy–lysosomal systems play a vital role in eliminating cellular detritus to maintain proteostasis and have been previously shown to be more robust in NMRs when compared with shorter-lived rodents. Using a 2-D PAGE proteomics approach, differential expression and phosphorylation levels of proteins involved in proteostasis networks were evaluated in the brains of NMRs in an age-dependent manner. We identified 9 proteins with significantly altered levels and/or phosphorylation states that have key roles involved in proteostasis networks. To further investigate the possible role that autophagy may play in maintaining cellular proteostasis, we examined aspects of the PI3K/Akt/mammalian target of rapamycin (mTOR) axis as well as levels of Beclin-1, LC3-I, and LC3-II in the brain of the NMR as a function of age. Together, these results show that NMRs maintain high levels of autophagy throughout the majority of their lifespan and may contribute to the extraordinary health span of these rodents. The potential of augmenting human health span via activating the proteostasis network will require further studies.Summary schematic diagram of expression proteomics and phosphoproteomics profiles of changes in proteins related to the ubiquitin–proteasomal system in the brain of the naked mole-rat (NMR) as a function of age. Proteins with significantly altered protein and/or phosphorylation levels with age in the NMR brain are labeled. Such proteins conceivably contribute to the extraordinary lifespan and health span of these rodents.Display Omitted
Keywords: Naked mole-rat; Proteomics; Phosphoproteomics; Aging; Proteostasis networks; mTOR;

NR2B-dependent cyclophilin D translocation suppresses the recovery of synaptic transmission after oxygen–glucose deprivation by Zhihua Zhang; Yongfu Wang; Shijun Yan; Fang Du; Shirley Shidu Yan (2225-2234).
N-methyl d-aspartate receptor (NMDA) subunit 2B (NR2B)-containing NMDA receptors and mitochondrial protein cyclophilin D (CypD) are well characterized in mediating neuronal death after ischemia, respectively. However, whether and how NR2B and CypD work together in mediating synaptic injury after ischemia remains elusive. Using an ex vivo ischemia model of oxygen–glucose deprivation (OGD) in hippocampal slices, we identified a NR2B-dependent mechanism for CypD translocation onto the mitochondrial inner membrane. CypD depletion (CypD null mice) prevented OGD-induced impairment in synaptic transmission recovery. Overexpression of neuronal CypD mice (CypD +) exacerbated OGD-induced loss of synaptic transmission. Inhibition of CypD-dependent mitochondrial permeability transition pore (mPTP) opening by cyclosporine A (CSA) attenuated ischemia-induced synaptic perturbation in CypD + and non-transgenic (non-Tg) mice. The treatment of antioxidant EUK134 to suppress mitochondrial oxidative stress rescued CypD-mediated synaptic dysfunction following OGD in CypD + slices. Furthermore, OGD provoked the interaction of CypD with P53, which was enhanced in slices overexpressing CypD but was diminished in CypD-null slices. Inhibition of p53 using a specific inhibitor of p53 (pifithrin-μ) attenuated the CypD/p53 interaction following OGD, along with a restored synaptic transmission in both non-Tg and CypD + hippocampal slices. Our results indicate that OGD-induced CypD translocation potentiates CypD/P53 interaction in a NR2B dependent manner, promoting oxidative stress and loss of synaptic transmission. We also evaluate a new ex vivo chronic OGD-induced ischemia model for studying the effect of oxidative stress on synaptic damage.
Keywords: NR2B; Mitochondria; OGD; Synaptic transmission; CypD; p53;