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BBA - Molecular Basis of Disease (v.1832, #1)

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-v).

MicroRNA-350 induces pathological heart hypertrophy by repressing both p38 and JNK pathways by Yuzhi Ge; Shujuan Pan; Di Guan; Hong Yin; Yong Fan; Jingjing Liu; Shuhua Zhang; Hongjie Zhang; Lili Feng; Yunxia Wang; Ruxiang Xu; James Q. Yin (pp. 1-10).

Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-350 in the cardiac setting is still unclear. The objective of this study is to determine whether miR-350 alone can induce pathological cardiac hypertrophy by repressing the SAPK pathway in cardiomyocytes. Here we report that miR-350 plays a key role in determining pathological cardiomyocyte hypertrophy and apoptosis. Comprehensive microarray profiling of miRs and qPCR showed that this unique miRNA was significantly up-regulated in rat hearts in response to late-stage transverse aortic constriction. Western blotting and luciferase assays confirmed that the target mRNAs of miR-350 are mitogen activated protein kinase (MAPK) 11/14 and MAPK8/9 gene transcripts. Gain-of-unction and loss-of-function approaches were used to investigate the functional roles of miR-350. The forced over-expression of miR-350 was sufficient to induce hypertrophy of cardiomyocytes through the posttranslational suppression of p38 and JNK protein synthesis. Moreover, miR-350 led to an increase in unphosphorylated NFATc3 and its nuclear translocation, resulting in the over-expression of pathological hypertrophy markers. As predicted, these effects could effectively be imitated by siR-JNK/p38 through the degeneration of p38 and JNK mRNAs. Conversely, antagomir-350 could lower the levels of miR-350, reverse the expression of target proteins and reduce cell size and apoptosis relative to the administration of mutant antagomir-350. Our data provide the first evidence that miR-350 is a critical regulator of pathological cardiac hypertrophy and apoptosis in rats.► MiR-350 over-expressed through p38 and JNK protein synthesis. ► MiR-350 led to an increase in unphosphorylated NFATc3. ► Antagomir-350 could lower the levels of miR-350. ► Antagomir-350 could reverse the expression of target proteins. ► MiR-350 is a critical regulator of pathological cardiac hypertrophy and apoptosis.

Keywords: Micro RNA; Cardiac hypertrophy; Apoptosis; Gene expression; Signaling pathway

Impact of obesity on IL-12 family gene expression in insulin responsive tissues by Heesun Nam; Bradley S. Ferguson; Jacqueline M. Stephens; Ron F. Morrison (pp. 11-19).

Mounting evidence has established a role for chronic inflammation in the development of obesity-induced insulin resistance, as genetic ablation of pro-inflammatory cytokines and chemokines elevated in obesity improves insulin signaling in vitro and in vivo. Recent evidence further highlights interleukin (IL)-12 family cytokines as prospective inflammatory mediators linking obesity to insulin resistance. In this study, we present empirical evidence demonstrating that IL-12 family related genes are expressed and regulated in insulin-responsive tissues under conditions of obesity. First, we report that respective mRNAs for each of the known members of this cytokine family are expressed within detectable ranges in WAT, skeletal muscle, liver and heart. Second, we show that these cytokines and their cognate receptors are divergently regulated with genetic obesity in a tissue-specific manner. Third, we demonstrate that select IL-12 family cytokines are regulated in WAT in a manner that is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity. Fourth, we report that respective mRNAs for IL-12 cytokines and receptors are also expressed and divergently regulated in cultured adipocytes under conditions of inflammatory stress. To our knowledge, this report is the first study to systemically evaluated mRNA expression of all IL-12 family cytokines and receptors in any tissue under conditions of obesity highlighting select family members as potential mediators linking excess nutrient intake to metabolic diseases such as insulin resistance, diabetes and heart disease.► IL-12 cytokines and receptors are regulated in insulin-responsive tissues by obesity. ► IL-12 family genes divergently regulated with obesity in a tissue-specific manner. ► IL-12 genes regulated in adipose tissue by developmental stage of obesity. ► IL-12 genes regulated in adipose tissue by inflammatory stress of obesity. ► IL-12 cytokines and receptors are regulated in adipocytes by inflammatory stress.

Keywords: Obesity; Adipose tissue; Adipocyte; Inflammation; Cytokine

Binding of methylene blue to a surface cleft inhibits the oligomerization and fibrillization of prion protein by Paola Cavaliere; Joan Torrent; Stephanie Prigent; Vincenzo Granata; Kris Pauwels; Annalisa Pastore; Human Rezaei; Adriana Zagari (pp. 20-28).

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.Display Omitted► MB is currently under study for neurodegenerative diseases. ► MB significantly affects the kinetics of PrP oligomerization. ► MB reduces the amount of PrP oligomer and completely suppresses fiber formation. ► NMR revealed that PrP hosts MB in a surface cleft. ► MB can be considered an anti-aggregation compound against proteinopathies.

Keywords: Abbreviations; PrP; prion protein; MB; methylene blue; wt-OvPrP; ovine recombinant wild-type full-length PrP (23–234); ΔOvPrP; ovine recombinant truncated PrP (103–234); MoPrP; murine recombinant full-length PrP (23–231); wt-HuPrP; wild-type human recombinant full-length PrP (23–231); AD; Alzheimer's diseaseAmyloidogenic protein; PrP oligomer; PrP fiber; Anti-prion agent; Neurodegenerative disease

Lysophosphatidic acid induces increased BACE1 expression and Aβ formation by Jing Shi; Yunzhou Dong; Mei-Zhen Cui; Xuemin Xu (pp. 29-38).

The abnormal production and accumulation of β-amyloid peptide (Aβ), which is produced from amyloid precursor protein (APP) by the sequential actions of β-secretase and γ-secretase, are thought to be the initial causative events in the development of Alzheimer's disease (AD). Accumulating evidence suggests that vascular factors play an important role in the pathogenesis of AD. Specifically, studies have suggested that one vascular factor in particular, oxidized low density lipoprotein (oxLDL), may play an important role in regulating Aβ formation in AD. However, the mechanism by which oxLDL modulates Aβ formation remains elusive. In this study, we report several new findings that provide biochemical evidence suggesting that the cardiovascular risk factor oxLDL may contribute to Alzheimer's disease by increasing Aβ production. First, we found that lysophosphatidic acid (LPA), the most bioactive component of oxLDL induces increased production of Aβ. Second, our data strongly indicate that LPA induces increased Aβ production via upregulating β-secretase expression. Third, our data strongly support the notion that different isoforms of protein kinase C (PKC) may play different roles in regulating APP processing. Specifically, most PKC members, such as PKCα, PKCβ, and PKCε, are implicated in regulating α-secretase-mediated APP processing; however, PKCδ, a member of the novel PKC subfamily, is involved in LPA-induced upregulation of β-secretase expression and Aβ production. These findings may contribute to a better understanding of the mechanisms by which the cardiovascular risk factor oxLDL is involved in Alzheimer's disease.► Evidence suggests that vascular factors play an important role in pathogenesis of AD. ► First, we found that LPA induces increased production of Aβ. ► Second, LPA induces increased Aβ production via upregulating β-secretase expression. ► Third, PKCδ is involved in LPA-induced β-secretase expression and Aβ production. ► Cardiovascular risk factors may contribute to AD by increasing Aβ production.

Keywords: Abbreviations; AD; Alzheimer's disease; NFT; neurofibrillary tangles; Aβ; β amyloid; APP; amyloid precursor protein; BACE1; β-site APP cleaving enzyme 1; oxLDL; oxidized low-density lipoprotein; CSF; cerebrospinal fluid; LPA; lysophosphatidic acid; CREB; cAMP response element-binding protein; APPsw; Swedish mutant APP; PS1wt; wild type presenilin 1; CM; conditioned medium; SRF; serum response element; CRE; cAMP-responsive element; PTX; pertussis toxin; wt; wild type; DN; dominant negative; lysoPLD; lysophospholipase DAlzheimer's disease; Lysophosphatidic acid; Oxidized LDL; β-Secretase; Beta-amyloid peptide; Amyloid precursor protein

Dietary curcumin counteracts extracellular transthyretin deposition: Insights on the mechanism of amyloid inhibition by Nelson Ferreira; Sónia A.O. Santos; Maria Rosário M. Domingues; Maria João Saraiva; Maria Rosário Almeida (pp. 39-45).

The transthyretin amyloidoses (ATTR) are devastating diseases characterized by progressive neuropathy and/or cardiomyopathy for which novel therapeutic strategies are needed. We have recently shown that curcumin (diferuloylmethane), the major bioactive polyphenol of turmeric, strongly suppresses TTR fibril formation in vitro, either by stabilization of TTR tetramer or by generating nonfibrillar small intermediates that are innocuous to cultured neuronal cells.In the present study, we aim to assess the effect of curcumin on TTR amyloidogenesis in vivo, using a well characterized mouse model for familial amyloidotic polyneuropathy (FAP). Mice were given 2% (w/w) dietary curcumin or control diet for a six week period. Curcumin supplementation resulted in micromolar steady-state levels in plasma as determined by LC/MS/MS. We show that curcumin binds selectively to the TTR thyroxine-binding sites of the tetramer over all the other plasma proteins.The effect on plasma TTR stability was determined by isoelectric focusing (IEF) and curcumin was found to significantly increase TTR tetramer resistance to dissociation. Most importantly, immunohistochemistry (IHC) analysis of mice tissues demonstrated that curcumin reduced TTR load in as much as 70% and lowered cytotoxicity associated with TTR aggregation by decreasing activation of death receptor Fas/CD95, endoplasmic reticulum (ER) chaperone BiP and 3-nitrotyrosine in tissues. Taken together, our results highlight the potential use of curcumin as a lead molecule for the prevention and treatment of TTR amyloidosis.► Dietary curcumin supplementation leads to stable levels of curcumin in mice plasma. ► Curcumin binds to plasma TTR and increases TTR resistance to dissociation. ► Curcumin decreases TTR deposition and associated biomarkers in tissues. ► Curcumin or derivatives can be used for TTR amyloidosis therapy.

Keywords: Transthyretin; Amyloid; Familial amyloidotic polyneuropathy; Curcumin

FTIR spectroscopy: A new valuable tool to classify the effects of polyphenolic compounds on cancer cells by Allison Derenne; Vincent Van Hemelryck; Delphine Lamoral-Theys; Robert Kiss; Erik Goormaghtigh (pp. 46-56).

Polyphenolic compounds are an important part of human diet and regular consumption of fruits, vegetables and tea is associated with reduced risk of cancer. Dietary polyphenols display a vast array of cellular effects but the large number of data published in the literature makes it difficult to determine the main mechanisms of action associated and to identify molecules with original mechanisms. Therefore, there is an increasing demand for more systemic approaches in order to obtain a global insight of the biochemical processes mediated by polyphenols. Here, we used Fourier transform infrared (FTIR) spectroscopy to analyze cancer cells exposed in vitro to 6 polyphenols: 3 natural well documented polyphenols (curcumin, epigallocatechin gallate (EGCG) and quercetin) and 3 synthetic molecules with a very closely related chemical structure. Statistical analyses on FTIR spectra allowed the comparison of global effects of the 6 compounds and evidenced some common or different features in the cell perturbations among natural and synthetic molecules. Interestingly, marked metabolic changes induced by polyphenols closely related from a chemical point of view were identified. Furthermore, many metabolic changes could be detected as early as after 2h incubation with the drugs.► FTIR spectroscopy provides a global signature of polyphenol effects on cancer cells. ► Curcumin, EGCG, and quercetin versus three synthetic (13a,13b,13c) polyphenols ► Mid-infrared spectroscopy of the human T98G glioblastoma cell line ► IR spectra provide an objective classifier of potential anticancer polyphenols.

Keywords: Abbreviations; IR; infrared; FTIR; Fourier transform infrared; h; hour; S/N; signal to noise; SD; standard deviation; PCA; principal component analysis; PC; principal component; MANOVA; multivariate analysis of variance; ESS; error sum of squarePolyphenol; IR spectroscopy; Cancer cell; IC; ₅₀

Overexpression of HSPA12B protects against cerebral ischemia/reperfusion injury via a PI3K/Akt-dependent mechanism by Yujie Ma; Chen Lu; Chuanfu Li; Rongrong Li; Yangyang Zhang; He Ma; Xiaojin Zhang; Zhengnian Ding; Li Liu (pp. 57-66).

Background and purpose: HSPA12B is a newly discovered member of the Hsp70 family proteins. This study investigated the effects of HSPA12B on focal cerebral ischemia/reperfusion (I/R) injury in mice. Methods: Transgenic mice overexpressing human HSPA12B (Tg) and wild-type littermates (WT) were subjected to 60min of middle cerebral artery occlusion to induce ischemia and followed by reperfusion (I/R). Neurological deficits, infarct volumes and neuronal death were examined at 6 and 24hrs after reperfusion. Blood–brain-barrier (BBB) integrity and activated cellular signaling were examined at 3hrs after reperfusion. Results: After cerebral I/R, Tg mice exhibited improvement in neurological deficits and decrease in infarct volumes, when compared with WT I/R mice. BBB integrity was significantly preserved in Tg mice following cerebral I/R. Tg mice also showed significant decreases in cell injury and apoptosis in the ischemic hemispheres. We observed that overexpression of HSPA12B activated PI3K/Akt signaling and suppressed JNK and p38 activation following cerebral I/R. Importantly, pharmacological inhibition of PI3K/Akt signaling abrogated the protection against cerebral I/R injury in Tg mice. Conclusions: The results demonstrate that HSPA12B protects the brains from focal cerebral I/R injury. The protective effect of HSPA12B is mediated though a PI3K/Akt-dependent mechanism. Our results suggest that HSPA12B may have a therapeutic potential against ischemic stroke.► HSPA12B protected brain from cerebral ischemia/reperfusion (I/R) injury. ► HSPA12B activated PI3K/Akt signaling in ischemic brains after cerebral I/R. ► Inhibition of PI3K abolished HSPA12B-induced protection against cerebral I/R. ► Thus, the neuroprotection of HSPA12B was mediated by PI3K/Akt activation.

Keywords: Abbreviations; I/R; ischemia/reperfusion; BBB; Blood–brain-barrier; HSPs; Heat shock proteins; HSPA12B; Heat shock protein A12B; PI3K/Akt; phosphoinositide 3-kinase/protein kinase B; TTC; 2,3,5-triphenyltetrazolium chloride; WM; WortmanninCerebral ischemia/reperfusion; Heat shock protein A12B (HSPA12B); PI3K/Akt signaling; Apoptosis; Neuroprotective agent

Is the mitochondrial outermembrane protein VDAC1 therapeutic target for Alzheimer's disease? by P. Hemachandra Reddy (pp. 67-75).

Mitochondrial dysfunction and synaptic damage have been described as early events in Alzheimer's disease (AD) pathogenesis. Recent research using AD postmortem brains, and AD mouse and cell models revealed that amyloid beta (Aβ) and tau hyperphosphorylation are involved in mitochondrial dysfunction and synaptic damage in AD. Further, recent research also revealed that the protein levels of mitochondrial outer membrane protein, voltage-dependent anion channel 1 (VDAC1), are elevated in the affected regions of AD postmortem brains and cortical tissues from APP transgenic mice. In addition, emerging research using AD postmortem brains and AD mouse models revealed that VDAC1 is linked to Aβ and phosphorylated tau, blocks the mitochondrial permeability transition (MPT) pores, disrupts the transport of mitochondrial proteins and metabolites, impairs gating of VDAC, and causes defects in oxidative phosphorylation, leading to mitochondrial dysfunction in AD neurons. The purpose of this article is to review research that has investigated the relationship between VDAC1 and the regulation of MPT pores in AD progression.► Mitochondrial dysfunction is an early event in Alzheimer's disease. ► VDAC1 protein levels are elevated in AD brains and cortical tissues from APP transgenic mice. ► VDAC1 is interacted with Aβ and phospho tau and disrupts the transport of metabolites. ► VDAC1+Aβ and Aβ+phospho tau complexes cause defects in OXPHOS in AD neurons.

Keywords: Alzheimer's disease; Amyloid beta; Amyloid precursor protein; Phoshorylated tau; Voltage-dependent anion channel; Mitochondrial dysfunction

Smad6 and Smad7 are co-regulated with hepcidin in mouse models of iron overload by Spasic Maja Vujić Spasić; Richard Sparla; Katarzyna Mleczko-Sanecka; Mary C. Migas; Katja Breitkopf-Heinlein; Steven Dooley; Sophie Vaulont; Robert E. Fleming; Martina U. Muckenthaler (pp. 76-84).

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe−/− and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.► Smad6, Smad7 and hepcidin are co-regulated in response to iron via Bmp/Smad-pathway. ► Hemochromatosis mouse models show inappropriately low hepcidin, Smad6 and Smad7 levels. ► Iron injections activate hepcidin, Smad6 and Smad7 mRNA levels despite the lack of Hfe. ► Bmp/Smad-independent signals may convey hepcidin, Smad6 and Smad7 response to iron.

Keywords: Smad6; Smad7; Hepcidin; Hemochromatosis; Iron; Bmp/Smad

Lysosomal enzymes are decreased in the kidney of diabetic rats by Giovani B. Peres; Maria A. Juliano; Simoes Manuel J. Simões; Yara M. Michelacci (pp. 85-95).

The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). The activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of β-glucuronidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. The main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy.► Lysosomal enzyme activities are decreased in type 1 DM rat kidney. ► Decrease was observed for cathepsins and glycosidases. ► Aryl sulfatases did not vary. ► Proximal convoluted tubules had thinner walls and decreased brush border. ► Cathepsin B was located in the brush border of tubular cells.

Keywords: Diabetes mellitus; Lysosome; Cathepsin; Glycosidase; Kidney; Tubular cells

CpG-ODN, the TLR9 agonist, attenuates myocardial ischemia/reperfusion injury: Involving activation of PI3K/Akt signaling by Zhijuan Cao; Danyang Ren; Tuanzhu Ha; Li Liu; Xiaohui Wang; John Kalbfleisch; Xiang Gao; Race Kao; David Williams; Chuanfu Li (pp. 96-104).

Toll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury.Male C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1h prior to myocardial ischemia (60min) followed by reperfusion. Untreated mice served as I/R control (n=10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14days.CpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection.CpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.► CpG-ODN administration attenuated ischemia/reperfusion-induced myocardial infarction. ► CpG-ODN improved cardiac function after myocardial reperfusion up to 14days. ► CpG-ODN administration increased Akt and GSK-3β phosphorylation in the myocardium. ► PI3K inhibition or Akt deficiency abolished CpG-ODN-induced cardioprotection.

Keywords: Myocardial I/R; TLR9; PI3K/Akt signaling; Apoptosis

Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein by Mariarosa A.B. Melone; Anna Calarco; Orsolina Petillo; Sabrina Margarucci; Luca Colucci-D'Amato; Umberto Galderisi; Guido Koverech; Gianfranco Peluso (pp. 105-113).

Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild‐type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.► Polyglutamine (polyQ) expansion in huntingtin (htt) protein causes Huntington's disease (HD). ► PolyQ-htt-induced alteration of EGFR trafficking affects cell migration and proliferation by EGF. ► Alteration in EGFR trafficking occcurs, at least in part, through inhibition of ERK signaling.

Keywords: Polyglutamine (polyQ); Huntingtin; Epidermal growth factor receptor (EGFR); Protein trafficking; Ubiquitination; Endosome

The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia by Matías Soncini; Fabio Santoro; Arantxa Gutierrez; Frige Gianmaria Frigè; Mauro Romanenghi; Oronza A. Botrugno; Isabella Pallavicini; PierGiuseppe Pelicci; Luciano Di Croce; Saverio Minucci (pp. 114-120).

Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor – decitabine (DAC) – in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects.► DAC induces apoptosis in AML cells and activates the apoptotic extrinsic pathway. ► DAC induces TRAIL expression in human AML cells. ► CpG-A rich region upstream of the TRAIL promoter is demethylated upon DAC treatment. ► CpG-A is a regulatory element whose activity can be modulated by methylation. ► CpG-A is hypermethylated in APL patients.

Keywords: Abbreviations; DAC; 5-aza-2′ Deoxycytidine or Decitabine; DNMT; DNA methyltransferase; HDAC; Histone deacetylaseAcute myeloid leukemia; PML–RAR; DNA methylation; Decitabine; TRAIL; Apoptosis

GLUT12 functions as a basal and insulin-independent glucose transporter in the heart by Amanda P. Waller; Michael George; Anuradha Kalyanasundaram; Chen Kang; Muthu Periasamy; Keli Hu; Véronique A. Lacombe (pp. 121-127).

Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.► Use of a biotinylated photolabeled assay in perfused myocardium and cardiac myocytes ► Insulin increased translocation of GLUT-4, but not -12, in the healthy myocardium ► Diabetes induced alteration in insulin-stimulated GLUT-4, but not -12, translocation. ► Active cell surface GLUT12 was increased in the diabetic myocardium.

Keywords: Abbreviations; GLUT; glucose transporters; Dx; diabetes; Con; control; L; labeled; UL; unlabeled; CASQ; calsequestrinDiabetes; GLUT translocation; Exofacial bis-mannose photolabeling; Myocytes; Perfused myocardium

Inhibition of CYP2E1 attenuates chronic alcohol intake-induced myocardial contractile dysfunction and apoptosis by Rong-Huai Zhang; Jian-Yuan Gao; Hai-Tao Guo; Glenda I. Scott; Anna R. Eason; Xiao-Ming Wang; Jun Ren (pp. 128-141).

Alcohol intake is associated with myocardial contractile dysfunction and apoptosis although the precise mechanism is unclear. This study was designed to examine the effect of the cytochrome P450 enzyme CYP2E1 inhibition on ethanol-induced cardiac dysfunction. Adult male mice were fed a 4% ethanol liquid or pair-fed control diet for 6weeks. Following 2weeks of diet feeding, a cohort of mice started to receive the CYP2E1 inhibitor diallyl sulfide (100mg/kg/d, i.p.) for the remaining feeding duration. Cardiac function was assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate CYP2E1, heme oxygenase-1 (HO-1), iNOS, the intracellular Ca2⁺ regulatory proteins sarco(endo)plasmic reticulum Ca2⁺-ATPase, Na⁺Ca2⁺ exchanger and phospholamban, pro-apoptotic protein cleaved caspase-3, Bax, c-Jun-NH₂-terminal kinase (JNK) and apoptosis signal-regulating kinase (ASK-1). Ethanol led to elevated levels of CYP2E1, iNOS and phospholamban, decreased levels of HO-1 and Na⁺Ca2⁺ exchanger, cardiac contractile and intracellular Ca2⁺ defects, cardiac fibrosis, overt O₂⁻ production, and apoptosis accompanied with increased phosphorylation of JNK and ASK-1, the effects were significantly attenuated or ablated by diallyl sulfide. Inhibitors of JNK and ASK-1 but not HO-1 inducer or iNOS inhibitor obliterated ethanol-induced cardiomyocyte contractile dysfunction, substantiating a role for JNK and ASK-1 signaling in ethanol-induced myocardial injury. Taken together, these findings suggest that ethanol metabolism through CYP2E1 may contribute to the pathogenesis of alcoholic cardiomyopathy including myocardial contractile dysfunction, oxidative stress and apoptosis, possibly through activation of JNK and ASK-1 signaling.► We examine the effect of CYP2E1 inhibition against alcoholic cardiomyopathy. ► Diallyl sulfide alleviates ethanol intake-induced cardiac defect and apoptosis. ► CYP2E1 inhibition inhibits oxidative stress, activation of ASK-1 and JNK.

Keywords: CYP2E1; Cardiac function; Apoptosis; JNK; ASK-1; Stress signaling

Uremic toxins inhibit renal metabolic capacity through interference with glucuronidation and mitochondrial respiration by H.A.M. Mutsaers; M.J.G. Wilmer; D. Reijnders; J. Jansen; P.H.H. van den Broek; M. Forkink; E. Schepers; G. Glorieux; R. Vanholder; L.P. van den Heuvel; J.G. Hoenderop; R. Masereeuw (pp. 142-150).

During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K_m of 12±2μM and a V_max of 76±3pmol/min/mg) and p-cresol (K_m of 33±13μM and a V_max of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase ( i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.► Human proximal tubule cell line to study renal pharmacokinetics. ► Uremic toxins decrease UDP-glucuronosyltransferase activity. ► Uremic toxins interact with mitochondria ( e.g. mitochondrial succinate dehydrogenase). ► Uremic toxins diminish the reserve capacity of the energy-generating OXPHOS system. ► Uremic toxins might play an important role in altering drug metabolism in patients with chronic kidney disease.

Keywords: Abbreviations; 7-OHC; 7-hydroxycoumarin; 7-OHCG; 7-hydroxycoumarin glucuronide; AA; antimycin A; ciPTEC; conditionally immortalized human renal proximal tubule epithelial cells; CKD; chronic kidney disease; CMPF; 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid; CRF; chronic renal failure; CYP; cytochrome p450; E: ETS; electron transport system; FCCP; p-trifluoromethoxy carbonyl cyanide phenyl hydrazone; FCS; fetal calf serum; GST; glutathione S-transferase; HA; hippuric acid; HEK293; human embryonic kidney cells; HPLC; high-performance liquid chromatography; I3A; indole-3-acetic acid; IS; indoxyl sulfate; KA; kynurenic acid; L; LEAK; M; medium; Mix; uremic toxin mix; MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NAD; ⁺; nicotinamide adenine dinucleotide; NAT; N-acetyltransferase; omy; oligomycin A; OAT; organic anion transporter; Ox; oxalate; OXPHOS; oxidative phosphorylation; pC; p-cresol; pCG; p-cresyl glucuronide; pCS; p-cresyl sulfate; PHA; phenylacetic acid; PHG; phenyl glucuronide; PHS; phenyl sulfate; PTEC; proximal tubule cells; Pu; putrescine; QA; quinolinic acid; R; ROUTINE; ROT; rotenone; ROX; residual oxygen consumption; SULT; sulfotransferase; UDPGA; UDP-glucuronic acid; UGT; UDP-glucuronosyltransferases; ZO-1; tight junction protein 1Uremic toxins; Chronic kidney disease; Drug metabolism; UDP-glucuronosyltransferases; Mitochondria

Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction by Yanju Ma; Shingo Semba; Md Rafiqul Islam Khan; Hiroki Bochimoto; Tsuyoshi Watanabe; Mikihiro Fujiya; Yutaka Kohgo; Yunpeng Liu; Takanobu Taniguchi (pp. 151-159).

Disruption of epithelial barrier function was identified as one of the pathologic mechanisms in inflammatory bowel diseases (IBD). Epithelial barrier consists of various intercellular junctions, in which the tight junction (TJ) is an important component. However, the regulatory mechanism of tight junction is still not clear. Here we examined the role of focal adhesion kinase (FAK) in the epithelial barrier function on Caco-2 monolayers using a specific FAK inhibitor, PF-573, 228 (PF-228). We found that the decrease of transepithelial resistance and the increase of paracellular permeability were accompanied with the inhibition of autophosphorylation of FAK by PF-228 treatment. In addition, PF-228 inhibited the FAK phosphorylation at Y576/577 on activation loop by Src, suggesting Src-dependent regulation of FAK in Caco-2 monolayers. In an ethanol-induced barrier injury model, PF-228 treatment also inhibited the recovery of transepithelial resistance as well as these phosphorylations of FAK. In a sucrose gradient ultracentrifugation, FAK co-localized with claudin-1, an element of the TJ complex, and they co-migrate after ethanol-induced barrier injury. Immunofluorescence imaging analysis revealed that PF-228 inhibited the FAK redistribution to the cell border and reassembly of TJ proteins in the recovery after ethanol-induced barrier injury. Finally, knockdown of FAK by siRNA resulted in the decrease of transepithelial resistance. These findings reveal that activation of FAK is necessary for maintaining and repairing epithelial barrier in Caco-2 cell monolayer via regulating TJ redistribution.► A role of focal adhesion kinase (FAK) in epithelial barrier function is explored. ► FAK activity is required in both maintenance and repair of epithelial barrier. ► FAK moves away from intercellular border to apical surface upon barrier injury. ► FAK co-localizes with tight junction to regulate its assembly/disassembly.

Keywords: Epithelial barrier; Focal adhesion kinase; Tight junction; ZO-1; Occludin

The hereditary spastic paraplegia protein strumpellin: Characterisation in neurons and of the effect of disease mutations on WASH complex assembly and function by Caroline Freeman; Matthew N.J. Seaman; Evan Reid (pp. 160-173).

Mutations in the gene encoding strumpellin cause autosomal dominant hereditary spastic paraplegia (HSP), in which there is degeneration of corticospinal tract axons. Strumpellin is a component of the WASH complex, an actin-regulating complex that is recruited to endosomes by interactions with the retromer complex. The WASH complex and its relationship to retromer have not been fully characterised in neurons, and the molecular pathological mechanism of strumpellin mutation is unclear. Here we demonstrate that the WASH complex assembles in the brain, where it interacts with retromer. Members of both complexes co-localise with each other and with endosomes in primary cortical neurons, and are present in somato-dendritic and axonal compartments. We show that strumpellin is not required for normal transferrin receptor traffic, but is required for the correct subcellular distribution of the β-2-adrenergic receptor. However, strumpellin disease mutations do not affect its incorporation into the WASH complex or its subcellular localisation, nor do they have a dominant effect on functions of the WASH complex, including regulation of endosomal tubulation, transferrin receptor traffic or β-2-adrenergic receptor localisation. Models of the WASH complex indicate that it contains a single strumpellin molecule, so in patients with strumpellin mutations, complexes containing wild-type and mutant strumpellin should be present in equal numbers. In most cell types this would provide sufficient functional WASH to allow normal cellular physiology. However, owing to the demands on membrane traffic imposed by their exceptionally long axons, we suggest that corticospinal neurons are especially vulnerable to reductions in functional WASH.► We studied the spastic paraplegia protein strumpellin, a component of WASH complex. ► The WASH complex assembles in neurons where it interacts with retromer on endosomes. ► WASH and retromer are in axons, the site of hereditary spastic paraplegia pathology. ► Strumpellin regulates the subcellular distribution of the β-2-adrenergic receptor. ► Disease mutants do not have a dominant effect on cellular functions of strumpellin.

Keywords: WASH complex; Retromer; Strumpellin; Hereditary spastic paraplegia; Axon degeneration; Sorting nexin

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease by María E. Solesio; Tracy A. Prime; Angela Logan; Michael P. Murphy; María del Mar Arroyo-Jimenez; Jordan Joaquín Jordán; María F. Galindo (pp. 174-182).

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50nM, 30min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.Display Omitted► Sublethal 6-OHDA concentration of 50μM, which is able to induce mitochondrial morphological alterations ► MitoQ abrogates 6-OHDA-induced mitochondrial fission. ► MitoQ reduces 6-OHDA-induced mitochondrial translocation of Drp.1 ► MitoQ reduces 6-OHDA-induced mitochondrial translocation of Bax.

Keywords: Mitochondrial dynamics; Fission; ROS; Bax; Drp1

Pathogenic role of HIF-1α in prostate hyperplasia in the presence of chronic inflammation by Hye-Jin Kim; Jong-Wan Park; Young-Suk Cho; Chung-Hyun Cho; Ji-Seon Kim; Hyun-Woo Shin; Doo Hyun Chung; Sang Jeong Kim; Yang-Sook Chun (pp. 183-194).

Benign prostatic hyperplasia (BPH) commonly occurs in older men with chronic prostatitis. Although BPH is frequently accompanied by inflammation, it is unclear whether inflammation underlies prostate enlargement. Recently, we reported that hypoxia‐inducible factor 1α (HIF-1α), which is known to be induced by proinflammatory cytokines, is involved in testosterone-induced prostate hyperplasia. Therefore, we hypothesized that cytokines secreted from infiltrated macrophages under inflammatory conditions stimulate prostate enlargement by up‐regulating HIF-1α. In the present study, we injected lipopolysaccharide (LPS) into rat prostates to mimic prostatitis and evaluated prostate hyperplasia 14days later. Epithelial cells of LPS-treated prostates were found to be highly proliferative and HIF-1α levels in prostate tissues to be elevated. When prostate epithelial cells were incubated in conditioned medium from macrophages activated with LPS, they robustly expressed HIF-1α, and under these conditions IL-1β, IL-6, and TNF-α cytokines were found to mediate HIF-1α induction. In addition, HIF-1α was found to enhance the expression of Twist, which initiates epithelial–mesenchymal transition (EMT). Furthermore, profound EMT features were observed in LPS-treated rat prostates, and the natural HIF-1α inhibitors ascorbate and curcumin were found to attenuate EMT and prostate hyperplasia both in vivo and in vitro. Based on these results, we propose that HIF-1α mediates prostate enlargement under inflammatory conditions, and we suggest that HIF-1α be viewed as a promising target for blocking the transition from prostatitis to BPH.► We induce prostate hyperplasia in rats by injecting LPS into prostates. ► Cytokines secreted from activated macrophages stimulate the HIF-1 signaling pathway. ► HIF-1 drives EMT in prostate epithelial cells. ► HIF-1 inhibitors block EMT and hyperplasia in LPS-treated prostates. ► This study will open a new concept about the pathogenesis of inflammatory BPH.

Keywords: Prostatic hyperplasia; LPS; Inflammation; HIF-1α; EMT; Cytokines

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin by Rooban B. Nahomi; Tomoko Oya-Ito; Ram H. Nagaraj (pp. 195-203).

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.► Lysine acetylation inhibits advanced glycation end product formation in α-crystallin. ► Lysine acetylation inhibits the loss of chaperone function by ascorbate glycation in α-crystallin. ► Lysine acetylation further improves the chaperone function of methylglyoxal-modified α-crystallin. ► Lysine acetylation improves the anti-apoptotic function of α-crystallin.

Keywords: Acetylation; α-Crystallin; Chaperone; Glycation; Apoptosis

S-adenosyl- L-homocysteine hydrolase and methylation disorders: Yeast as a model system by Oksana Tehlivets; Nermina Malanovic; Myriam Visram; Tea Pavkov-Keller; Walter Keller (pp. 204-215).

S-adenosyl- L-methionine (AdoMet)-dependent methylation is central to the regulation of many biological processes: more than 50 AdoMet-dependent methyltransferases methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids. Common to all AdoMet-dependent methyltransferase reactions is the release of the strong product inhibitor S-adenosyl- L-homocysteine (AdoHcy), as a by-product of the reaction. S-adenosyl- L-homocysteine hydrolase is the only eukaryotic enzyme capable of reversible AdoHcy hydrolysis to adenosine and homocysteine and, thus, relief from AdoHcy inhibition. Impaired S-adenosyl- L-homocysteine hydrolase activity in humans results in AdoHcy accumulation and severe pathological consequences. Hyperhomocysteinemia, which is characterized by elevated levels of homocysteine in blood, also exhibits a similar phenotype of AdoHcy accumulation due to the reversal of the direction of the S-adenosyl- L-homocysteine hydrolase reaction. Inhibition of S-adenosyl- L-homocysteine hydrolase is also linked to antiviral effects. In this review the advantages of yeast as an experimental system to understand pathologies associated with AdoHcy accumulation will be discussed.► AdoHcy is a potent product inhibitor of AdoMet-dependent methyltransferases. ► AdoHcy accumulates in hyperhomocysteinemia. ► Yeast is an advantageous system to understand AdoHcy toxicity. ► Lipid metabolism is deregulated in response to AdoHcy accumulation.

Keywords: AdoMet; AdoHcy; Homocysteine; S; -adenosyl-; L; -homocysteine hydrolase

Regulation of mitochondrial F_oF₁ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977bp deletion of mitochondrial DNA by Yu-Ting Wu; Hsin-Chen Lee; Chen-Chung Liao; Yau-Huei Wei (pp. 216-227).

Sirt3, a mitochondrial NAD⁺-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F_oF₁ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F_oF₁ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F_oF₁ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F_oF₁ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F_oF₁ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ≅85% of mtDNA with 4977bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F_oF₁ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5–10μM of menadione impaired the Sirt3-mediated deacetylation and activation on F_oF₁ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F_oF₁ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.► We demonstrated a role of Sirt3 in the regulation of acetylation and activity of mitochondrial F_oF₁ATPase. ► The OSCP subunit of F_oF₁ATPase was identified as a new target of Sirt3. ► ROS overproduction led to a decline in the expression level of Sirt3 and the impairment of F_oF₁ATPase. ► Oxidative stress is involved in the Sirt3 deficiency in human cells harboring the 4977bp deletion of mtDNA.

Keywords: Energy metabolism; F; _o; F; ₁; ATPase; Lysine acetylation; Mitochondrial disease; Oxidative stress; Sirt3

Altered feeding differentially regulates circadian rhythms and energy metabolism in liver and muscle of rats by Jane Reznick; Elaine Preston; Donna L. Wilks; Susan M. Beale; Nigel Turner; Gregory J. Cooney (pp. 228-238).

Energy metabolism follows a diurnal pattern responding to the light/dark cycle and food availability. This study investigated the impact of restricting feeding to the daylight hours and feeding a high fat diet on circadian clock ( bmal1, dbp, tef and e4bp4) and metabolic ( pepck, fas, ucp3, pdk4) gene expression and markers of energy metabolism in muscle and liver of rats. The results show that in chow-fed rats switched to daylight feeding, the peak diurnal expression of genes in liver was shifted by 6–12h while expression of these genes in muscle remained in a similar phase to rats feeding ad libitum. High fat feeding during the daylight hours had limited effect on clock gene expression in liver or muscle but shifted the peak expression of metabolic genes ( pepck, fas) in liver by 6–12h. The differential effects of daylight feeding on gene and protein expression in muscle and liver were accompanied by an 8% reduction in whole body energy expenditure, a 20–30% increased glycogen content during the light phase in muscle of day-fed rats and increased adipose tissue deposition per gram food consumed. These data demonstrate that a mismatch of feeding and light/dark cycle disrupts tissue metabolism in muscle with significant consequences for whole body energy homeostasis.► Muscle and liver in rats respond differently to limiting feeding to daylight hours. ► Altered muscle glycogen storage and decreased energy expenditure in day-fed rats. ► Diurnal gene expression and glycogen content shift in liver to mimic food intake. ► Disrupted energy metabolism in day-fed rats predisposes to fat deposition. ► Differences in the response to day-feeding with chow and high fat diet

Keywords: Circadian rhythm; Energy metabolism; Liver; Muscle

PPARγ ligand treatment inhibits cardiac inflammatory mediators induced by infection with different lethality strains of Trypanosoma cruzi by Federico Penas; Gerardo A. Mirkin; Eugenia Hovsepian; Ágata Cevey; Roberto Caccuri; María Elena Sales; Nora B. Goren (pp. 239-248).

Trypanosoma cruzi ( T. cruzi), the etiological agent of Chagas' disease, causes cardiac alterations in the host. Although the main clinical manifestations arise during the chronic stage, the mechanisms leading to heart damage develop early during infection. In fact, an intense inflammatory response is observed from acute stage of infection. Recently, peroxisome proliferator-activated receptors (PPARs) have attracted research interest due to their participation in the modulation of inflammation. In this work we addressed the role of 15-Deoxy-∆^12,14 ProstaglandinJ2 (15dPGJ2), a PPARγ natural ligand in the regulation of inflammatory mediators, in acute and chronic experimental mouse models of Chagas' disease with the RA and K98 T. cruzi strains, respectively. This work demonstrates that 15dPGJ2 treatment inhibits the expression and activity of inducible nitric oxide synthase (NOS2) as well as TNF-α and IL-6 mRNA levels. Also, expression and activity of metalloproteinases 2 (MMP-2) and 9 (MMP9) were inhibited by 15dPGJ2. Moreover GW9662, a specific PPARγ antagonist, revealed the participation of other signaling pathways since, in GW9662 presence, 15dPJG2 had a partial effect on the inhibition of inflammatory parameters in the acute model of infection. Accordingly, NF-κB activation was demonstrated, assessing p65 nuclear translocation in the hearts of infected mice with both T. cruzi strains. Such effect was inhibited after 15dPGJ2 treatment. Our findings support the concept that in vivo PPARγ and NF-κB pathways are implicated in the inhibitory effects of 15dPGJ2 on inflammatory mediators at different times depending on whether the infection is caused by the lethal or non-lethal T. cruzi strain.► 15dPGJ2 modulates inflammatory mediators in the hearts of mice infected with T. cruzi. ► NOS2, MMPs, TNF-α and IL-6 decrease in the hearts of 15dPGJ2-treated mice. ► PPARγ-dependent and ‐independent (NF-κB) pathways are involved in 15dPGJ2 effect.

Keywords: PPARγ; Trypanosoma cruzi; Heart; Inflammation

Transport and distribution of⁴⁵Ca2⁺ in the perfused rat liver and the influence of adjuvant-induced arthritis by Karina Sayuri Utsunomiya; Luiz Guilherme Scaliante; Adelar Bracht; Emy Luiza Ishii-Iwamoto (pp. 249-262).

The purpose of the present work was to investigate Ca2⁺ transport and distribution under the conditions of the intact rat liver in health and disease (adjuvant-induced arthritis). The multiple-indicator dilution technique was used with the simultaneous injection of⁴⁵Ca2⁺ and indicators into the portal vein under defined conditions and analysis of the outflow profiles by means of a space-distributed variable transit time model. The best description of the⁴⁵Ca2⁺ outflow profiles corresponds to a model that assumes rapid distribution of⁴⁵Ca2⁺ between the vascular space and the cell surface and a slower transfer into the hepatocytes. In kinetic terms two distinct cellular pools were distinguishable, the cytosol and the endoplasmic reticulum. The concentration of Ca2⁺ in the cytosol was much lower than in the vascular space and in the endoplasmic reticulum. The most prominent modification observed in the livers of arthritic rats was the increased Ca2⁺ concentration in the hormone-sensitive cellular pool. Furthermore, reduced rates of Ca2⁺ influx and efflux between the hormone-sensitive cellular pool and the cytosolic space were also detected in combination with a significantly reduced expression of the sarco-endoplasmic reticulum Ca2⁺-ATPase (SERCA2) protein. All these observations mean that in livers from arthritic rats more time is required to replenish the hormone sensitive Ca2⁺ stores.► Ca2⁺ transport in the liver was examined by multiple-indicator dilution technique. ► A four-pool model for Ca2⁺ distribution was devised for steady state conditions. ► Arthritis alters Ca2⁺ exchange in the hormone-sensitive cellular pool. ► Modifications in Ca2⁺ homeostasis caused by diseases can be kinetically evaluated.

Keywords: Multiple-indicator dilution technique; ⁴⁵; Ca; 2; ⁺; space-distribution; Rat liver; Norepinephrine; Adjuvant-induced arthritis

Excitotoxic stimulation downregulates the ubiquitin–proteasome system through activation of NMDA receptors in cultured hippocampal neurons by Margarida V. Caldeira; Michele Curcio; Graciano Leal; Ivan L. Salazar; Miranda Mele; Ana Rita A. Santos; Carlos V. Melo; Paulo Pereira; Lorella M.T. Canzoniero; Carlos B. Duarte (pp. 263-274).

Overactivation of glutamate receptors contributes to neuronal damage (excitotoxicity) in ischemic stroke but the detailed mechanisms are not fully elucidated. Brain ischemia is also characterized by an impairment of the activity of the proteasome, one of the major proteolytic systems in neurons. We found that excitotoxic stimulation with glutamate rapidly decreases ATP levels and the proteasome activity, and induces the disassembly of the 26S proteasome in cultured rat hippocampal neurons. Downregulation of the proteasome activity, leading to an accumulation of ubiquitinated proteins, was mediated by calcium entry through NMDA receptors and was only observed in the nuclear fraction. Furthermore, excitotoxicity-induced proteasome inhibition was partially sensitive to cathepsin-L inhibition and was specifically induced by activation of extrasynaptic NMDA receptors. Oxygen and glucose deprivation induced neuronal death and downregulated the activity of the proteasome by a mechanism dependent on the activation of NMDA receptors. Since deubiquitinating enzymes may regulate proteins half-life by counteracting ubiquitination, we also analyzed how their activity is regulated under excitotoxic conditions. Glutamate stimulation decreased the total deubiquitinase activity in hippocampal neurons, but was without effect on the activity of Uch-L1, showing that not all deubiquitinases are affected. These results indicate that excitotoxic stimulation with glutamate has multiple effects on the ubiquitin–proteasome system which may contribute to the demise process in brain ischemia and in other neurological disorders.► Glutamate-induced excitotoxicity decreases proteasome activity, particularly in nucleus. ► Extrasynaptic NMDA receptors contribute to the downregulation of proteasome activity. ► OGD impairs proteasome activity by mechanisms dependent on NMDA receptor activation. ► Glutamate-induced excitotoxicity evokes 26S proteasome disassembly. ► Proteasome inhibition induces hippocampal neuronal death.

Keywords: UPS; Proteasome; Excitotoxicity; Glutamate; NMDA receptors; OGD

The tyrosine phosphatase TC48 interacts with and inactivates the oncogenic fusion protein BCR-Abl but not cellular Abl by Aninda Mitra; Kotagiri Sasikumar; B.V.V. Parthasaradhi; Vegesna Radha (pp. 275-284).

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms — TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.► BCR-Abl interacts with and is a substrate of tyrosine phosphatase, TC48. ► TC48 mediated dephosphorylation of BCR-Abl at Y1127 and Y1294 causes its inactivation. ► c-Abl, or its active variants are not dephosphorylated and inactivated by TC48. ► TC48 expression inhibits growth and enhances STI571 sensitivity of K562 cells.

Keywords: Abl; Abelson; PTP; protein tyrosine phosphatase; TCPTP; T-cell PTP; CML; chronic myelogenous leukemia; BCR; break point cluster gene

Evaluation of the pathogenicity of GJB3 and GJB6 variants associated with nonsyndromic hearing loss by Se-Kyung Oh; Soo-Young Choi; Song Hee Yu; Kyu-Yup Lee; Jeong Hwa Hong; Sung Won Hur; Sang Jeong Kim; Chang-Jin Jeon; Un-Kyung Kim (pp. 285-291).

A number of genes responsible for hearing loss are related to ion recycling and homeostasis in the inner ear. Connexins (Cx26 encoded by GJB2, Cx31 encoded by GJB3 and Cx30 encoded by GJB6) are core components of gap junctions in the inner ear. Gap junctions are intercellular communication channels and important factors that are associated with hearing loss. To date, a molecular genetics study of GJB3 and GJB6 as a causative gene for hearing loss has not been performed in Korea. This study was therefore performed to elucidate the genetic characteristics of Korean patients with nonsyndromic sensorineural hearing loss and to determine the pathological mechanism of hearing loss by analyzing the intercellular communication function of Cx30 and Cx31 variants. Sequencing analysis of the GJB3 and GJB6 genes in our population revealed a total of nine variants, including four novel variants in the two genes. Three of the novel variants (Cx31-p.V27M, Cx31-p.V43M and Cx-30-p.I248V) and two previously reported variants (Cx31-p.V84I and Cx30-p.A40V) were selected for functional studies using a pathogenicity prediction program and assessed for whether the mutations were located in a conserved region of the protein. The results of biochemical and ionic coupling tests showed that both the Cx31-p.V27M and Cx31-p.V84I variants did not function normally when each was expressed as a heterozygote with the wild-type Cx31. This study demonstrated that two variants of Cx31 were pathogenic mutations with deleterious effect. This information will be valuable in understanding the pathogenic role of GJB3 and GJB6 mutations associated with hearing loss.► Pathological evaluation of hearing loss of Cx30 and Cx31 by biochemical and ionic coupling tests. ► Nine variants including four novel variants in two genes. ► A pathogenic effect of both Cx31-p.V27M and Cx31-p.V84I variants.

Keywords: Hearing loss; Gap junction; Connexin; Variant; Functional study

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BBA - Molecular Basis of Disease (v.1832, #1)