BBA - Molecular Basis of Disease (v.1862, #9)

Ceramides are a diverse group of sphingolipids that play important roles in many biological processes. Acid ceramidase (AC) is one key enzyme that regulates ceramide metabolism. Early research on AC focused on the fact that it is the enzyme deficient in the rare genetic disorder, Farber Lipogranulomatosis. Recent research has revealed that deficiency of the same enzyme is responsible for a rare form of spinal muscular atrophy associated with myoclonic epilepsy (SMA-PME). Due to their diverse role in biology, accumulation of ceramides also has been implicated in the pathobiology of many other common diseases, including infectious lung diseases, diabetes, cancers and others. This has revealed the potential of AC as a therapy for many of these diseases. This review will focus on the biology of AC and the potential role of this enzyme in the treatment of human disease.
Keywords: Ceramide; Sphingolipids; Enzymes; Lysosomal storage diseases; Cell signaling;

Frequent inactivation of MCC/CTNNBIP1 and overexpression of phospho-beta-cateninY654 are associated with breast carcinoma: Clinical and prognostic significance by Nupur Mukherjee; Hemantika Dasgupta; Rittwika Bhattacharya; Debolina Pal; Rituparna Roy; Saimul Islam; Neyaz Alam; Jaydip Biswas; Anup Roy; Susanta Roychoudhury; Chinmay Kumar Panda (1472-1484).
Transcriptional activation of β-catenin is a hallmark of Wnt/β-catenin pathway activation. The MCC (Mutated in colorectal cancers) and CTNNBIP1 (catenin, beta interacting protein 1) are two candidate genes which inhibit the transcriptional activity of nuclear β-catenin. The importance of MCC and CTNNBIP1 in breast cancer (BC) development has not yet been studied in detail. For this reason, in present study, the alterations (deletion/methylation/mutation/expression) of MCC and CTNNBIP1 were analyzed in BC of Indian patients (N  = 120) followed by expression/mutation analysis of β-catenin. Then transcriptional activity of β-catenin was checked by expression analysis of its target genes (EGFR, C-MYC and CCND1) in the same set of samples. Frequent methylation (44–45%) than deletion (20–32%) with overall alterations of 52–55% was observed in MCC/CTNNBIP1 in the BC samples. The alterations of MCC/CTNNBIP1 showed significant correlation with increased nuclear β-catenin/p-β-cateninY654 expression. Also, a significant correlation was seen between nuclear β-catenin expression and overexpression of its target genes like EGFR, MYC and CCND1 in the BC samples (P  < 0.0001). An upregulation of MCC and CTNNBIP1 expression by 5-Aza-2′-deoxycytidine treatment of MCF7 and MDA-MB-231 cell lines lead to downregulation of β-catenin and its target genes. The expression of nuclear p-β-cateninY654, EGFR, MYC and CCND1 were significantly high in TNBC (Triple negative BC) and Her2 + compared to Luminal A/B + subtypes. The TNBC patients in stage III/IV having reduced expression of MCC in the tumors showed poor prognosis. Thus, our data suggests that inactivation of MCC/CTNNBIP1 could be an important event in activation of β-catenin mediated transcription of target genes in BC.
Keywords: Breast cancer; MCC/CTNNBIP1; Nuclear β-catenin; p-β-cateninY654; EGFR/MYC/CCND1; Breast cancer subtypes;

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), have been increasingly recognized as important regulators of energy homeostasis. The orexigenic agouti-related peptide (AgRP), initially identified as an endogenous antagonist for both neural MCRs, has been suggested to be a biased agonist of MC4R independent of its antagonizing effects. In the present study, we sought to determine the potential of AgRP to regulate the activation of intracellular kinases, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), AKT and AMP-activated protein kinase (AMPK), through neural MCRs. We showed that AgRP acted as a biased agonist in human MC3R (hMC3R), decreasing cAMP activity of constitutively active mutant (F347A) hMC3R but stimulating ERK1/2 activation in both wide type and F347A hMC3Rs. AgRP-stimulated ERK1/2 phosphorylation through MC3R was abolished by protein kinase A (PKA) inhibitor H-89 but not Rp-cAMPS, whereas AgRP-initiated ERK1/2 activation through MC4R was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Both NDP-MSH and AgRP treatment induced significant AKT phosphorylation in GT1-7 cells but not in MC3R- or MC4R-transfected HEK293T cells. The phosphorylated AMPK levels in both GT1-7 cells and HERK293T cells transfected with neural MCRs were significantly decreased upon stimulation with NDP-MSH but not with AgRP. In summary, we provided novel data for AgRP-initiated multiple intracellular signaling pathways, demonstrating biased agonism of AgRP in both neural MCRs, leading to a better understanding of neural MCR pharmacology.
Keywords: Melanocortin-3 receptor; Melanocortin-4 receptor; Agouti-related peptide; Biased signaling;

Impact of Dyrk1A level on alcohol metabolism by Marjorie Renon; Béatrice Legrand; Etienne Blanc; Fabrice Daubigney; Cindy Bokobza; Marie Mortreux; Jean-Louis Paul; Jean-Maurice Delabar; Hélène Rouach; Karine Andreau; Nathalie Janel (1495-1503).
Alcoholic liver diseases arise from complex phenotypes involving many genetic factors. It is quite common to find hyperhomocysteinemia in chronic alcoholic liver diseases, mainly due to deregulation of hepatic homocysteine metabolism. Dyrk1A, involved in homocysteine metabolism at different crossroads, is decreased in liver of hyperhomocysteinemic mice. Here, we hypothesized that Dyrk1A contributes to alcohol-induced hepatic impairment in mice. Control, hyperhomocysteinemic and mice overexpressing Dyrk1A were fed using a Lieber-DeCarli liquid diet with or without ethanol (5% v/v ethanol) for one month, and liver histological examination and liver biochemical function tests were performed. Plasma alanine aminotransferase and homocysteine levels were significantly decreased in mice overexpressing Dyrk1A compared to control mice with or without alcohol administration. On the contrary, the mean plasma alanine aminotransferase and homocysteine levels were significantly higher in hyperhomocysteinemic mice than that of control mice after alcohol administration. Paraoxonase 1 and CYP2E1, two phase I xenobiotic metabolizing enzymes, were found increased in the three groups of mice after alcohol administration. However, NQO1, a phase II enzyme, was only found increased in hyperhomocysteinemic mice after alcohol exposure, suggesting a greater effect of alcohol in liver of hyperhomocysteinemic mice. We observed positive correlations between hepatic alcohol dehydrogenase activity, Dyrk1A and ADH4 protein levels. Importantly, a deleterious effect of alcohol consumption on hepatic Dyrk1A protein level was found. Our study reveals on the one hand a role of Dyrk1A in ethanol metabolism and on the other hand a deleterious effect of alcohol administration on hepatic Dyrk1A level.
Keywords: Ethanol; Alcohol dehydrogenase; Liver; Mice; Hyperhomocysteinemia;

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues.
Keywords: Uridine-cytidine kinase; Neuroblastoma; Nucleoside analogues; Metabolism; Cytotoxicity;

Nuclear speckles are detention centers for transcripts containing expanded CAG repeats by Martyna O. Urbanek; Magdalena Jazurek; Pawel M. Switonski; Grzegorz Figura; Wlodzimierz J. Krzyzosiak (1513-1520).
The human genetic disorders caused by CAG repeat expansions in the translated sequences of various genes are called polyglutamine (polyQ) diseases because of the cellular “toxicity” of the mutant proteins. The contribution of mutant transcripts to the pathogenesis of these diseases is supported by several observations obtained from cellular models of these disorders. Here, we show that the common feature of cell lines modeling polyQ diseases is the formation of nuclear CAG RNA foci. We performed qualitative and quantitative analyses of these foci in numerous cellular models endogenously and exogenously expressing mutant transcripts by fluorescence in situ hybridization (FISH). We compared the CAG RNA foci of polyQ diseases with the CUG foci of myotonic dystrophy type 1 and found substantial differences in their number and morphology. Smaller differences within the polyQ disease group were also revealed and included a positive correlation between the foci number and the CAG repeat length. We show that expanded CAA repeats, also encoding glutamine, did not trigger RNA foci formation and foci formation is independent of the presence of mutant polyglutamine protein. Using FISH combined with immunofluorescence, we demonstrated partial co-localization of CAG repeat foci with MBNL1 alternative splicing factor, which explains the mild deregulation of MBNL1-dependent genes. We also showed that foci reside within nuclear speckles in diverse cell types: fibroblasts, lymphoblasts, iPS cells and neuronal progenitors and remain dependent on integrity of these nuclear structures.
Keywords: Polyglutamine diseases; RNA toxicity; CAG foci; CUG foci; Splicing speckles;

Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function by Melanie M. Pleiss; Pradoldej Sompol; Susan D. Kraner; Hafiz Mohmmad Abdul; Jennifer L. Furman; Rodney P. Guttmann; Donna M. Wilcock; Peter T. Nelson; Christopher M. Norris (1521-1532).
Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45–48 kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury.
Keywords: Calcineurin; Proteolysis; Astrocytes; Alzheimer's disease; Microinfarct;

Novel effects of sphingosylphosphorylcholine on invasion of breast cancer: Involvement of matrix metalloproteinase-3 secretion leading to WNT activation by Hyun Ji Kim; Gyeoung Jin Kang; Eun Ji Kim; Mi Kyung Park; Hyun Jung Byun; Seungyoon Nam; Ho Lee; Chang Hoon Lee (1533-1543).
Sphingosylphosphorylcholine (SPC) participates in several cellular processes including metastasis. SPC induces keratin reorganization and regulates the viscoelasticity of metastatic cancer cells including PANC-1 cancer cells leading to enhanced migration and invasion. The role of SPC and the relevant mechanism in invasion of breast cell are as yet unknown. SPC dose-dependently induces invasion of breast cancer cells or breast immortalized cells. Reverse transcription polymerase chain reaction and Western blot analyses of MCF10A and ZR-75-1 cells indicated that SPC induces expression and secretion of matrix metalloproteinase-3 (MMP3). From online KMPLOT, relapse free survival is high in patients having low MMP3 expressed basal breast cancer (n  = 581, p  = 0.032). UK370106 (MMP3 inhibitor) or gene silencing of MMP3 markedly inhibited the SPC-induced invasion of MCF10A cells. An extracellular signal-regulated kinase (ERK) inhibitor, PD98059, significantly suppressed the secretion and the gelatinolytic activity of MMP3, and invasion in MCF10A cells. Over-expression of ERK1 and ERK2 promoted both the expression and secretion of MMP3. In contrast, gene silencing of ERK1 and ERK2 attenuated the secretion of MMP3 in MCF10A cells. The effects of SPC-induced MMP3 secretion on β-catenin and TCF/lymphoid enhancer factor (LEF) promoter activity were examined since MMP3 indirectly activates canonical Wnt signaling. SPC induced translocation of β-catenin to nucleus and increased TCF/LEF promoter activity. These events were suppressed by UK370106 or PD98059. Wnt inhibitor, FH535 inhibited SPC-induced MMP3 secretion and invasion.Taken together, these results suggest that SPC induces MMP3 expression and secretion via ERK leading to Wnt activation.Display Omitted
Keywords: Sphingosylphosphorylcholine; MMP3; UK370106; Wnt; FH535; Breast cancer;

Tyrosine phosphorylation of RACK1 triggers cardiomyocyte hypertrophy by regulating the interaction between p300 and GATA4 by Hidetoshi Suzuki; Yasufumi Katanasaka; Yoichi Sunagawa; Yusuke Miyazaki; Masafumi Funamoto; Hiromichi Wada; Koji Hasegawa; Tatsuya Morimoto (1544-1557).
The zinc finger protein GATA4 is a transcription factor involved in cardiomyocyte hypertrophy. It forms a functional complex with the intrinsic histone acetyltransferase (HAT) p300. The HAT activity of p300 is required for the acetylation and transcriptional activity of GATA4, as well as for cardiomyocyte hypertrophy and the development of heart failure. In the present study, we have identified Receptor for Activated Protein Kinase C1 (RACK1) as a novel GATA4-binding protein using tandem affinity purification and mass spectrometry analyses. We found that exogenous RACK1 repressed phenylephrine (PE)-induced hypertrophic responses, such as myofibrillar organization, increased cell size, and hypertrophy-associated gene transcription, in cultured cardiomyocytes. RACK1 physically interacted with GATA4 and the overexpression of RACK1 reduced PE-induced formation of the p300/GATA4 complex and the acetylation and DNA binding activity of GATA4. In response to hypertrophic stimulation in cultured cardiomyocytes and in the hearts of hypertensive heart disease model rats, the tyrosine phosphorylation of RACK1 was increased, and the binding between GATA4 and RACK1 was reduced. In addition, the tyrosine phosphorylation of RACK1 was required for the disruption of the RACK1/GATA4 complex and for the formation of the p300/GATA4 complex. These findings demonstrate that RACK1 is involved in p300/GATA4-dependent hypertrophic responses in cardiomyocytes and is a promising therapeutic target for heart failure.
Keywords: Cardiac hypertrophy; Acetyltransferase; Phosphotyrosine; p300; GATA4; RACK1;

Mammalian ataxin-2 modulates translation control at the pre-initiation complex via PI3K/mTOR and is induced by starvation by Isabel Lastres-Becker; David Nonis; Florian Eich; Michael Klinkenberg; Myriam Gorospe; Peter Kötter; Fabrice A.C. Klein; Nancy Kedersha; Georg Auburger (1558-1569).
Ataxin-2 is a cytoplasmic protein, product of the ATXN2 gene, whose deficiency leads to obesity, while its gain-of-function leads to neural atrophy. Ataxin-2 affects RNA homeostasis, but its effects are unclear. Here, immunofluorescence analysis suggested that ataxin-2 associates with 48S pre-initiation components at stress granules in neurons and mouse embryonic fibroblasts, but is not essential for stress granule formation. Coimmunoprecipitation analysis showed associations of ataxin-2 with initiation factors, which were concentrated at monosome fractions of polysome gradients like ataxin-2, unlike its known interactor PABP. Mouse embryonic fibroblasts lacking ataxin-2 showed increased phosphorylation of translation modulators 4E-BP1 and ribosomal protein S6 through the PI3K-mTOR pathways. Indeed, human neuroblastoma cells after trophic deprivation showed a strong induction of ATXN2 transcript via mTOR inhibition. Our results support the notion that ataxin-2 is a nutritional stress-inducible modulator of mRNA translation at the pre-initiation complex.Display Omitted
Keywords: Diabetes mellitus; Spinocerebellar Ataxia type 2; Amyotrophic lateral sclerosis; TORC1; mRNA translation;

Mannose 6-phosphate-dependent targeting of lysosomal enzymes is required for normal craniofacial and dental development by Till Koehne; Sandra Markmann; Michaela Schweizer; Nicole Muschol; Reinhard E. Friedrich; Christian Hagel; Markus Glatzel; Bärbel Kahl-Nieke; Michael Amling; Thorsten Schinke; Thomas Braulke (1570-1580).
Mucolipidosis II (MLII) is a severe systemic genetic disorder caused by defects in mannose 6-phosphate-dependent targeting of multiple lysosomal hydrolases and subsequent lysosomal accumulation of non-degraded material. MLII patients exhibit marked facial coarseness and gingival overgrowth soon after birth, accompanied with delayed tooth eruption and dental infections. To examine the pathomechanisms of early craniofacial and dental abnormalities, we analyzed mice with an MLII patient mutation that mimic the clinical and biochemical symptoms of MLII patients. The mouse data were compared with clinical and histological data of gingiva and teeth from MLII patients. Here, we report that progressive thickening and porosity of calvarial and mandibular bones, accompanied by elevated bone loss due to 2-fold higher number of osteoclasts cause the characteristic craniofacial phenotype in MLII. The analysis of postnatal tooth development by microcomputed tomography imaging and histology revealed normal dentin and enamel formation, and increased cementum thickness accompanied with accumulation of storage material in cementoblasts of MLII mice. Massive accumulation of storage material in subepithelial cells as well as disorganization of collagen fibrils led to gingival hypertrophy. Electron and immunofluorescence microscopy, together with 35S-sulfate incorporation experiments revealed the accumulation of non-degraded material, non-esterified cholesterol and glycosaminoglycans in gingival fibroblasts, which was accompanied by missorting of various lysosomal proteins (α-fucosidase 1, cathepsin L and Z, Npc2, α-l-iduronidase). Our study shows that MLII mice closely mimic the craniofacial and dental phenotype of MLII patients and reveals the critical role of mannose 6-phosphate-dependent targeting of lysosomal proteins for alveolar bone, cementum and gingiva homeostasis.
Keywords: Cementoblasts; Craniofacial anomalies; Gingival hypertrophy; Lysosomes; Mucolipidosis II; Osteoclastogenesis;

Mixed – Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states by Siobhan M. Craige; Michaella M. Reif; Shashi Kant (1581-1586).
Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases.
Keywords: Signal transduction; Kinase; Inflammation; Metabolism; Insulin resistance; Disease; Mixed-lineage kinase;

Aldehyde dehydrogenase 2 deficiency blunts compensatory cardiac hypertrophy through modulating Akt phosphorylation early after transverse aorta constriction in mice by Guang Xia; Fan Fan; Ming Liu; Shijun Wang; Jian Wu; Cheng Shen; Shasha Han; Cong Wang; Jianguo Jia; Yunzeng Zou; Kai Hu; Junbo Ge; Aijun Sun (1587-1593).
This study was designed to examine the impact of mitochondrial aldehyde dehydrogenase 2 (ALDH2) on transverse aorta constriction (TAC)-induced cardiac hypertrophy and related molecular mechanisms using an ALDH2 knockout (ALDH2 −/−) murine model.Male wild-type and ALDH2 −/− mice were subjected to TAC or sham operation (n = 6–8 for each group). After two weeks, cardiac function was assessed by echocardiography and hemodynamic measurements. Myocardial phosphorylated and total PI3K, the catalytic subunit of PI3Ks (p110α and p110γ), Akt, and total PTEN levels were detected by Western blotting. Cardiomyocytes were stretched for 6 h in vitro in the presence or absence of Alda-1 (a small-molecule activator of ALDH2) prior to assessment of phosphorylated PI3K, Akt and total PTEN expressions by Western blot.Heart to body weight ratio and left ventricular posterior wall thickness as well as the cross-sectional area of cardiomyocyte were significantly lower in ALDH2 −/− mice than in wild-type mice following TAC. Western blot analysis showed p110γ was upregulated post TAC in both wild-type mice and ALDH2 −/− mice, phosphorylation of Akt was disrupted, PTEN expression was upregulated in ALDH2 −/− mice post TAC while phosphorylated PI3K, p110α and p110γ expression was similar between ALDH2 −/− and wild-type mice post TAC. In vitro, phosphorylation of Akt was significantly accentuated and PTEN expression was reduced while PI3K phosphorylation remained unchanged in stretched cardiomyocytes pretreated by Alda-1 compared to stretched cardiomyocytes treated by saline.Our results show that ALDH2 deficiency attenuates compensatory cardiac hypertrophy through regulating Akt but not PI3K phosphorylation early after TAC in mice.
Keywords: ALDH2; Compensatory cardiac hypertrophy; Transverse aorta constriction; PI3K; Akt; PTEN;

Regulation of angiogenin expression and epithelial-mesenchymal transition by HIF-1α signaling in hypoxic retinal pigment epithelial cells by Kairan Lai; Chenqi Luo; Xiaobo Zhang; Panpan Ye; Yidong Zhang; Jiliang He; Ke Yao (1594-1607).
Choroidal neovascularization (CNV) is a major cause of vision loss in many retinal diseases. Hypoxia is determined to be a key inducer of CNV and hypoxia-inducible factor-1 (HIF-1) is an important transcription factor. Epithelial-mesenchymal transition (EMT) and the synthesis of proangiogenic cytokines make great contributions to the development of CNV. In the present study, the role of HIF-1α signaling in the regulation of angiogenin (ANG) expression and EMT in hypoxic retinal pigment epithelial cells was investigated. A significant elevation expression of ANG expression level in a mouse model of laser-induced CNV was demonstrated. In a hypoxic model of ARPE-19, an increased expression level of ANG and induction of EMT accompanied with stabilization and nucleus translocation of HIF-1α. Blockage of HIF-1α signaling resulted in inhibition of high expression of ANG and EMT features. The direct interaction between HIF-1α and ANG promoter region was identified by ChIP-qPCR. The association of RNase 4 mRNA level with HIF-1α signaling was also clarified in APRE-19. Moreover, the exogenous ANG translocated into the nucleus, enhanced 45S rRNA transcription, promoted cell proliferation and tube formation in human retinal microvascular endothelial cells. In conclusion, the hypoxic conditions regulate the expression of ANG and EMT via an activation of HIF-1α signaling. It provides molecular evidence for potential therapy strategies of treating CNV.
Keywords: Angiogenin; RNase 4; Epithelial-mesenchymal transition; Hypoxia-inducible factor-1; Choroidal neovascularization;

Oxidative and nitrative stress and pro-inflammatory cytokines in Mucopolysaccharidosis type II patients: effect of long-term enzyme replacement therapy and relation with glycosaminoglycan accumulation by Carlos Eduardo Diaz Jacques; Bruna Donida; Caroline P. Mescka; Daiane G.B. Rodrigues; Desirèe P. Marchetti; Fernanda H. Bitencourt; Maira G. Burin; Carolina F.M. de Souza; Roberto Giugliani; Carmen Regla Vargas (1608-1616).
Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a deficient activity of iduronate-2-sulfatase, leading to abnormal accumulation of glycosaminoglycans (GAG). The main treatment for MPS II is enzyme replacement therapy (ERT). Previous studies described potential benefits of six months of ERT against oxidative stress in patients. Thus, the aim of this study was to investigate oxidative, nitrative and inflammatory biomarkers in MPS II patients submitted to long term ERT. It were analyzed urine and blood samples from patients on ERT (mean time: 5.2 years) and healthy controls. Patients presented increased levels of lipid peroxidation, assessed by urinary 15-F2t-isoprostane and plasmatic thiobarbituric acid-reactive substances. Concerning to protein damage, urinary di-tyrosine (di-Tyr) was increased in patients; however, sulfhydryl and carbonyl groups in plasma were not altered. It were also verified increased levels of urinary nitrate + nitrite and plasmatic nitric oxide (NO) in MPS II patients. Pro-inflammatory cytokines IL-1β and TNF-α were increased in treated patients. GAG levels were correlated to di-Tyr and nitrate + nitrite. Furthermore, IL-1β was positively correlated with TNF-α and NO. Contrastingly, we did not observed alterations in erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, in reduced glutathione content and in the plasmatic antioxidant capacity. Although some parameters were still altered in MPS II patients, these results may suggest a protective role of long-term ERT against oxidative stress, especially upon oxidative damage to protein and enzymatic and non-enzymatic defenses. Moreover, the redox imbalance observed in treated patients seems to be GAG- and pro-inflammatory cytokine-related.
Keywords: Mucopolysaccharidosis type II; Oxidative stress; Pro-inflammatory cytokines; Enzyme replacement therapy; Reactive oxygen species; Reactive nitrogen species;

Are circulating microRNAs peripheral biomarkers for Alzheimer's disease? by Subodh Kumar; P. Hemachandra Reddy (1617-1627).
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss, multiple cognitive abnormalities and intellectual impairments. Currently, there are no drugs or agents that can delay and/or prevent the progression of disease in elderly individuals, and there are no peripheral biomarkers that can detect AD early in its pathogenesis. Research has focused on identifying biomarkers for AD so that treatment can be begun as soon as possible in order to restrict or prevent intellectual impairments, memory loss, and other cognitive abnormalities that are associated with the disease. One such potential biomarker is microRNAs that are found in circulatory biofluids, such as blood and blood components, serum and plasma. Blood and blood components are primary sources where miRNAs are released in either cell-free form and then bind to protein components, or are in an encapsulated form with microvesicle particles. Exosomal miRNAs are known to be stable in biofluids and can be detected by high throughput techniques, like microarray and RNA sequencing. In AD brain, enriched miRNAs encapsulated with exosomes crosses the blood brain barrier and secreted in the CSF and blood circulations. This review summarizes recent studies that have identified miRNAs in the blood, serum, plasma, exosomes, cerebral spinal fluids, and extracellular fluids as potential biomarkers of AD. Recent research has revealed only six miRNAs – miR-9, miR-125b, miR-146a, miR-181c, let-7g-5p, and miR-191-5p – that were reported by multiple investigators. Some studies analyzed the diagnostic potential of these six miRNAs through receiver operating curve analysis which indicates the significant area-under-curve values in different biofluid samples. miR-191-5p was found to have the maximum area-under-curve value (0.95) only in plasma and serum samples while smaller area-under-curve values were found for miR-125, miR-181c, miR-191-5p, miR-146a, and miR-9. This article shortlisted the promising miRNA candidates and discussed their diagnostic properties and cellular functions in order to search for potential biomarker for AD.
Keywords: Alzheimer's disease; Circulatory microRNA; Biomarker; Serum; Cerebral spinal fluid; CSF; Plasma;

Treatment with polyamine oxidase inhibitor reduces microglial activation and limits vascular injury in ischemic retinopathy by C. Patel; Z. Xu; E. Shosha; J. Xing; R. Lucas; R.W. Caldwell; R.B. Caldwell; S.P. Narayanan (1628-1639).
Retinal vascular injury is a major cause of vision impairment in ischemic retinopathies. Insults such as hyperoxia, oxidative stress and inflammation contribute to this pathology. Previously, we showed that hyperoxia-induced retinal neurodegeneration is associated with increased polyamine oxidation. Here, we are studying the involvement of polyamine oxidases in hyperoxia-induced injury and death of retinal vascular endothelial cells. New-born C57BL6/J mice were exposed to hyperoxia (70% O2) from postnatal day (P) 7 to 12 and were treated with the polyamine oxidase inhibitor MDL 72527 or vehicle starting at P6. Mice were sacrificed after different durations of hyperoxia and their retinas were analyzed to determine the effects on vascular injury, microglial cell activation, and inflammatory cytokine profiling. The results of this analysis showed that MDL 72527 treatment significantly reduced hyperoxia-induced retinal vascular injury and enhanced vascular sprouting as compared with the vehicle controls. These protective effects were correlated with significant decreases in microglial activation as well as levels of inflammatory cytokines and chemokines. In order to model the effects of polyamine oxidation in causing microglial activation in vitro, studies were performed using rat brain microvascular endothelial cells treated with conditioned-medium from rat retinal microglia stimulated with hydrogen peroxide. Conditioned-medium from activated microglial cultures induced cell stress signals and cell death in microvascular endothelial cells. These studies demonstrate the involvement of polyamine oxidases in hyperoxia-induced retinal vascular injury and retinal inflammation in ischemic retinopathy, through mechanisms involving cross-talk between endothelial cells and resident retinal microglia.Display Omitted

Iron overload inhibits osteogenic commitment and differentiation of mesenchymal stem cells via the induction of ferritin by Enikő Balogh; Emese Tolnai; Béla Nagy; Béla Nagy; György Balla; József Balla; Viktória Jeney (1640-1649).
Osteogenic differentiation of multipotent mesenchymal stem cells (MSCs) plays a crucial role in bone remodeling. Numerous studies have described the deleterious effect of iron overload on bone density and microarchitecture. Excess iron decreases osteoblast activity, leading to impaired extracellular matrix (ECM) mineralization. Additionally, iron overload facilitates osteoclast differentiation and bone resorption. These processes contribute to iron overload-associated bone loss. In this study we investigated the effect of iron on osteogenic differentiation of human bone marrow MSCs (BMSCs), the third player in bone remodeling.We induced osteogenic differentiation of BMSCs in the presence or absence of iron (0–50 μmol/L) and examined ECM mineralization, Ca content of the ECM, mRNA and protein expressions of the osteogenic transcription factor runt-related transcription factor 2 (Runx2), and its targets osteocalcin (OCN) and alkaline phosphatase (ALP). Iron dose-dependently attenuated ECM mineralization and decreased the expressions of Runx2 and OCN. Iron accomplished complete inhibition of osteogenic differentiation of BMSCs at 50 μmol/L concentration. We demonstrated that in response to iron BMSCs upregulated the expression of ferritin. Administration of exogenous ferritin mimicked the anti-osteogenic effect of iron, and blocked the upregulation of Runx2, OCN and ALP. Iron overload in mice was associated with elevated ferritin and decreased Runx2 mRNA levels in compact bone osteoprogenitor cells. The inhibitory effect of iron is specific toward osteogenic differentiation of MSCs as neither chondrogenesis nor adipogenesis were influenced by excess iron. We concluded that iron and ferritin specifically inhibit osteogenic commitment and differentiation of BMSCs both in vitro and in vivo.Display Omitted
Keywords: Human mesenchymal stem cell; Osteogenic differentiation; Iron; Ferritin; Runx2;

Metabolite mapping reveals severe widespread perturbation of multiple metabolic processes in Huntington's disease human brain by Stefano Patassini; Paul Begley; Jingshu Xu; Stephanie J. Church; Suzanne J. Reid; Eric H. Kim; Maurice A. Curtis; Mike Dragunow; Henry J. Waldvogel; Russell G. Snell; Richard D. Unwin; Richard L.M. Faull; Garth J.S. Cooper (1650-1662).
Huntington's disease (HD) is a genetically-mediated neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein (Htt) through lengthening of its polyglutamine tract, thus initiating a cascade that ultimately leads to premature death. However, neurodegeneration typically manifests in HD only in middle age, and mechanisms linking the causative mutation to brain disease are poorly understood. Brain metabolism is severely perturbed in HD, and some studies have indicated a potential role for mutant Htt as a driver of these metabolic aberrations. Here, our objective was to determine the effects of HD on brain metabolism by measuring levels of polar metabolites in regions known to undergo varying degrees of damage. We performed gas-chromatography/mass spectrometry-based metabolomic analyses in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine matched controls. In each patient, we measured metabolite content in representative tissue-samples from eleven brain regions that display varying degrees of damage in HD, thus identifying the presence and abundance of 63 different metabolites from several molecular classes, including carbohydrates, amino acids, nucleosides, and neurotransmitters. Robust alterations in regional brain-metabolite abundances were observed in HD patients: these included changes in levels of small molecules that play important roles as intermediates in the tricarboxylic-acid and urea cycles, and amino-acid metabolism. Our findings point to widespread disruption of brain metabolism and indicate a complex phenotype beyond the gradient of neuropathologic damage observed in HD brain.Display Omitted
Keywords: Neurodegenerative disease; Huntington's disease; Polyol pathway; Brain urea metabolism; Metabolic brain disease; Inositol pathway;

Modulation of microglia polarization dynamics during diabetic retinopathy in db/db mice by Ana I. Arroba; Elena Alcalde-Estevez; Marta García-Ramírez; Daniele Cazzoni; Pedro de la Villa; Elena M. Sánchez-Fernández; Carmen Ortiz Mellet; José M. García Fernández; Cristina Hernández; Rafael Simó; Ángela M. Valverde (1663-1674).
Retinal diseases linked to inflammation are often accompanied by macrophage/microglial cells activation. However, the dynamics between M1 (pro-inflammatory) and M2 (anti-inflammatory) polarization of microglia during diabetic retinopathy (DR) has not been investigated and it might be therapeutically useful. We assessed microglia polarization in retinas from db/db mice and human diabetic donors and also the microglia-mediated anti-inflammatory effects of the bicyclic nojirimycin derivative (1R)-1-dodecylsulfinyl-5N,6O-oxomethylidenenojirimycin (R-DS-ONJ). Visual function in mice was evaluated by electroretinogram (ERG). Expression of pro- and anti-inflammatory markers in the retina was analyzed by immunofluorescence, Western-blot and quantitative real-time PCR. Lipopolysaccharide (LPS)-mediated polarization profile was studied in Bv.2 microglial cells in the absence or presence of anti-inflammatory cytokines (IL4/IL13) or R-DS-ONJ. At 5 weeks of age, reduced ERG amplitude values of rod and mixed waves were detected in db/db compared to db/+ mice that correlated with elevated circulating endotoxemia and pro-inflammatory cytokines. At this early stage of DR, the marker of activated microglia Iba-1 co-localized with the M2 marker arginase-1 in the retina. Conversely, in retinas from 8 weeks old db/db mice Iba-1-colocalized with active caspase-1, a key component of the inflammasome, reflecting an opposite pattern of microglia polarization. Markers of activated microglia were detected in retinas of diabetic donors. Treatment of Bv.2 cells with LPS and IL4/IL13 or R-DS-ONJ switched the M1 response towards M2. In retinal explants from db/db mice, R-DS-ONJ induced a M2 response. In conclusion, the modulation of microglia polarization dynamics towards a M2 status at early stages of DR offers novel therapeutic interventions.
Keywords: Diabetic retinopathy; Microglia; Inflammation;

Metabolic signatures of Huntington's disease (HD): 1H NMR analysis of the polar metabolome in post-mortem human brain by Stewart F. Graham; Praveen K. Kumar; Trent Bjorndahl; BeomSoo Han; Ali Yilmaz; Eric Sherman; Ray O. Bahado-Singh; David Wishart; David Mann; Brian D. Green (1675-1684).
Huntington's disease (HD) is an autosomal neurodegenerative disorder affecting approximately 5–10 persons per 100,000 worldwide. The pathophysiology of HD is not fully understood but the age of onset is known to be highly dependent on the number of CAG triplet repeats in the huntingtin gene. Using 1H NMR spectroscopy this study biochemically profiled 39 brain metabolites in post-mortem striatum (n = 14) and frontal lobe (n = 14) from HD sufferers and controls (n = 28). Striatum metabolites were more perturbed with 15 significantly affected in HD cases, compared with only 4 in frontal lobe (p < 0.05; q < 0.3). The metabolite which changed most overall was urea which decreased 3.25-fold in striatum (p < 0.01). Four metabolites were consistently affected in both brain regions. These included the neurotransmitter precursors tyrosine and l-phenylalanine which were significantly depleted by 1.55–1.58-fold and 1.48–1.54-fold in striatum and frontal lobe, respectively (p = 0.02–0.03). They also included l-leucine which was reduced 1.54–1.69-fold (p = 0.04–0.09) and myo-inositol which was increased 1.26–1.37-fold (p < 0.01). Logistic regression analyses performed with MetaboAnalyst demonstrated that data obtained from striatum produced models which were profoundly more sensitive and specific than those produced from frontal lobe. The brain metabolite changes uncovered in this first 1H NMR investigation of human HD offer new insights into the disease pathophysiology. Further investigations of striatal metabolite disturbances are clearly warranted.
Keywords: Huntington's disease; Metabolomics; 1H NMR; Brain; Metabolites;

The lncRNA MALAT1, acting through HIF-1α stabilization, enhances arsenite-induced glycolysis in human hepatic L-02 cells by Fei Luo; Xinlu Liu; Min Ling; Lu Lu; Le Shi; Xiaolin Lu; Jun Li; Aihua Zhang; Qizhan Liu (1685-1695).
Accelerated glycolysis, a common process in tumor cells called the Warburg effect, is associated with various biological phenomena. However, the role of glycolysis induced by arsenite, a well-established human carcinogen, is unknown. Long non-coding RNAs (lncRNAs) act as regulators in various cancers, but how lncRNAs regulate glucose metabolism remains largely unexplored. We have found that, in human hepatic epithelial (L − 02) cells, arsenite increases lactate production; glucose consumption; and expression of glycolysis-related genes, including HK-2, Eno-1, and Glut-4. In L-02 cells exposed to arsenite, the lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and hypoxia inducible factors (HIFs)-α, the transcriptional regulators of cellular response to hypoxia, are over-expressed. In addition, HIF-1α, not HIF-2α, is involved in arsenite-induced glycolysis, and MALAT1 enhances arsenite-induced glycolysis. Although MALAT1 regulates HIF-α and promotes arsenite-induced glycolysis, MALAT1 promotes glycolysis through HIF-1α, not HIF-2α. Moreover, arsenite-increased MALAT1 enhances the disassociation of Von Hippel–Lindau (VHL) tumor suppressor from HIF-1α, alleviating VHL-mediated ubiquitination of HIF-1α, which causes accumulation of HIF-1α. In sum, these findings indicate that MALAT1, acting through HIF-1α stabilization, is a mediator that enhances glycolysis induced by arsenite. These results provide a link between the induction of lncRNAs and the glycolysis in cells exposed to arsenite, and thus establish a previously unknown mechanism for arsenite-induced hepatotoxicity.A schematic illustration of a model depicting a mechanism by which MALAT1 regulates HIF-1α to promote glycolysis in L-02 cells exposed to arsenite.Based on our data, we propose a model depicting a role of MALAT1 in the regulation of arsenite-enhanced glycolysis. In L-02 cells, MALAT1 is induced by arsenite. With arsenite exposure, MALAT1 blocks HIF-1α hydroxylation and then disrupts the HIF-1α-VHL interaction, stabilizes HIF-1α and increases expression of glycolytic enzymes, such as HK-2, Eno-1, and Glut-4 thereby promoting glycolysis; HIF-1α enhances arsenite-induced glycolysis may through p53 and NF-κB. In summary, in L-02 cells exposed to arsenite, MALAT1 is involved in the Warburg effect.Display Omitted
Keywords: lncRNAs; HIFs; Arsenite; Glycolysis;

Rhizoma Coptidis alkaloids alleviate hyperlipidemia in B6 mice by modulating gut microbiota and bile acid pathways by Kai He; Yinran Hu; Hang Ma; Zongyao Zou; Yubo Xiao; Yong Yang; Min Feng; Xuegang Li; Xiaoli Ye (1696-1709).
It is hypothesized that Rhizoma Coptidis (RC) alkaloids exert their hypolipidemic effects primarily by targeting the gastrointestinal tract and liver. Thus, this study was conducted to evaluate the antihyperlipidemic mechanisms of RC alkaloids (at a daily dose of 140 mg/kg for 35 days) in high-fat and high-cholesterol induced hyperlipidemic B6 mice. After treatment, serum lipid parameters were determined, the expression of lipid metabolism related genes and pathways such as the sterol regulatory element binding proteins (SREBPs) and bile acid signaling in mice were also investigated. Meanwhile, Illumina sequencing was used to investigate the differences in gut microbiota of B6 mice. The results indicated that RC alkaloids reduced the body weight gain and serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), total bile acids (TBA) and lipopolysaccharide of B6 mice. Liver fat deposition and epididymal adipose cell size were also deceased in therapy group. RC alkaloids feeding significantly promoted the abundance of Sporobacter termitidis, Alcaligenes faecalis, Akkermansia muciniphila in the gut of mice, whereas, the abundance of Escherichia coli, Desulfovibrio C21_c20, Parabacteroides distasonis was suppressed. The observed antihyperlipidemic effects of RC alkaloids can also be attributed to their action as agonists of FXR and TGR5, activators for SREBP2, LDLR, UCP2 and CYP7A1, inhibitors of HMGCR, TXNIP, TLR4 and JNK. Therefore, this study expands current knowledge on hypolipidemic mechanisms of RC alkaloids and presents new evidence supporting a key role for RC alkaloids as regulators of lipid homeostasis by modulation gut microbiota and hepatic lipid metabolism.Display Omitted
Keywords: Rhizoma Coptidis; Alkaloids; B6 mice; Antihyperlipidemic; Gut microbiota; Mechanisms;

Cyclooxygenase-2 expression in hepatocytes attenuates non-alcoholic steatohepatitis and liver fibrosis in mice by Omar Motiño; Noelia Agra; Rocío Brea Contreras; Marina Domínguez-Moreno; Carmelo García-Monzón; Javier Vargas-Castrillón; Cristina E. Carnovale; Lisardo Boscá; Marta Casado; Rafael Mayoral; M. Pilar Valdecantos; Ángela M. Valverde; Daniel E. Francés; Paloma Martín-Sanz (1710-1723).
Cyclooxygenase-2 (COX-2) is involved in different liver diseases but little is known about the significance of COX-2 in the development and progression of non-alcoholic steatohepatitis (NASH). This study was designed to elucidate the role of COX-2 expression in hepatocytes in the pathogenesis of steatohepatitis and hepatic fibrosis. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either fed methionine-and-choline deficient (MCD) diet to establish an experimental non-alcoholic steatohepatitis (NASH) model or injected with carbon tetrachloride (CCl4) to induce liver fibrosis. In our animal model, hCOX-2-Tg mice fed MCD diet showed lower grades of steatosis, ballooning and inflammation than Wt mice, in part by reduced recruitment and infiltration of hepatic macrophages, with a corresponding decrease in serum levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice showed a significant attenuation of the MCD diet-induced increase in oxidative stress and hepatic apoptosis observed in Wt mice. Even more, hCOX-2-Tg mice treated with CCl4 had significantly lower stages of fibrosis and less hepatic content of collagen, hydroxyproline and pro-fibrogenic markers than Wt controls. Collectively, our data indicates that constitutive hepatocyte COX-2 expression ameliorates NASH and liver fibrosis development in mice by reducing inflammation, oxidative stress and apoptosis and by modulating activation of hepatic stellate cells, respectively, suggesting a possible protective role for COX-2 induction in NASH/NAFLD progression.
Keywords: COX-2; Liver; Steatohepatitis; Inflammation; Fibrosis;

Osteogenic differentiation of human lens epithelial cells might contribute to lens calcification by Enikő Balogh; Andrea Tóth; Emese Tolnai; Tímea Bodó; Emese Bányai; Dóra Júlia Szabó; Goran Petrovski; Viktória Jeney (1724-1731).
Calcification of the human lens has been described in senile cataracts and in young patients with congenital cataract or chronic uveitis. Lens calcification is also a major complication of cataract surgery and plays a role in the opacification of intraocular lenses. A cell-mediated process has been suggested in the background of lens calcification, but so far the exact mechanism remained unexplored. Lens calcification shares remarkable similarities with vascular calcification; in both pathological processes hydroxyapatite accumulates in the soft tissue. Vascular calcification is a regulated, cell-mediated process in which vascular cells undergo osteogenic differentiation. Our objective was to investigate whether human lens epithelial cells (HuLECs) can undergo osteogenic transition in vitro, and whether this process contributes to lens calcification. We used inorganic phosphate (Pi) and Ca to stimulate osteogenic differentiation of HuLECs. Osteogenic stimuli (2.5 mmol/L Pi and 1.2 mmol/L Ca) induced extracellular matrix mineralization and Ca deposition in HuLECs with the critical involvement of active Pi uptake. Osteogenic stimuli almost doubled mRNA expressions of osteo-/chondrogenic transcription factors Runx2 and Sox9, which was accompanied by a 1.9-fold increase in Runx2 and a 5.5-fold increase in Sox9 protein expressions. Osteogenic stimuli induced mRNA and protein expressions of alkaline phosphatase and osteocalcin in HuLEC. Ca content was higher in human cataractous lenses, compared to non-cataractous controls (n = 10). Osteocalcin, an osteoblast-specific protein, was expressed in 2 out of 10 cataractous lenses. We conclude that osteogenic stimuli induce osteogenic differentiation of HuLECs and propose that this mechanism might play a role in lens calcification.Display Omitted
Keywords: Human lens epithelial cells; Osteogenic differentiation; Calcification; Cataract; Aging disease;

The RNA binding KH domain of Spoonbill depletes pathogenic non-coding spinocerebellar ataxia 8 transcripts and suppresses neurodegeneration in Drosophila by Bipin K. Tripathi; Satya Surabhi; Pradeep K. Bhaskar; Ashim Mukherjee; Mousumi Mutsuddi (1732-1741).
Spinocerebellar ataxia 8 (SCA8) pathogenesis is a resultant of gain-of-function machinery that primarily results at the RNA level. It has been reported that expanded non-coding CTG trinucleotide repeat in the ATXN8OS transcripts leads to SCA8 coupled neurodegeneration. Targeted depletion of pathogenic SCA8 transcripts is a viable therapeutic approach. In this report we have focused on the suppression of toxic RNA gain-of-function associated with SCA8. We report suppression of SCA8 associated neurodegeneration by KH RNA binding domain of Spoonbill. KH domain suppresses pathogenic SCA8 associated phenotype in adult flies. Ectopic expression of KH domain leads to massive reduction in the number and size of SCA8 RNA foci. We show that Spoonbill interacts with toxic SCA8 transcripts via its KH domain and promotes its depletion. Till date, no attempts have been made for therapeutic intervention of SCA8 pathogenesis. Further characterization of Spoonbill KH domain may aid us in designing peptide based therapeutics for SCA8 associated neurodegeneration.
Keywords: Spinocerebellar Ataxia 8; Neurodegeneration; RNA gain-of-function; Drosophila melanogaster; Spoonbill; KH domain;

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are related neurodegenerative disorders which are characterized by a rapid decline in cognitive and motor functions, and short survival. Both syndromes may be present within the same family or even in the same person. The genetic findings for both diseases also support the existence of a continuum, with mutations in the same genes being found in patients with ALS, FTD or FTD/ALS. Little is known about the molecular mechanisms underlying the differences in mutations of the same protein causing either ALS or FTD. Here, we shed light on 348 ALS and FTD missense mutations in 14 genes focusing on genic intolerance and protein stability based on available 3D structures. Using EvoTol, we prioritized the disease-causing genes and their domain. The most intolerant genes predicted by EvoTol are SQSTM1 and OPTN which are involved in protein homeostasis. Further, using ENCoM (Elastic Network Contact Model) that predicts stability based on vibrational entropy, we predicted that most of the missense mutations with destabilizing energies are in the structural regions that control the protein–protein interaction, and only a few mutations affect protein folding. We found a trend that energy changes are higher for ALS compared to FTD mutations. The stability of the ALS mutants correlated well with the duration of disease progression as compared to FTD–ALS mutants. This study provides a comprehensive understanding of the mechanism of ALS and illustrates the significance of structure-energy based studies in differentiating ALS and FTD mutations.Display Omitted
Keywords: Amyotrophic lateral sclerosis; Frontotemporal dementia; Mutation; Protein stability; Gene intolerance;

Aberrant hippocampal Atp8a1 levels are associated with altered synaptic strength, electrical activity, and autistic-like behavior by Daniel J. Kerr; Alexandra Marsillo; Sara R. Guariglia; Tatyana Budylin; Rodina Sadek; Silvia Menkes; Abha Chauhan; Guang Y. Wen; Daniel P. McCloskey; Andrzej Wieraszko; Probal Banerjee (1755-1765).
Type IV ATPases are putative aminophospholipid translocases (APLTs), more commonly known as flippases. A pronounced induction of the flippase Atp8a1 was observed in post-mortem tissue homogenates from the hippocampus and temporal lobe of juvenile autistic subjects compared to age-matched controls. In order to simulate the human data, C57BL/6 mice were allowed to develop after intra-hippocampal injection of recombinant lentivirus expressing Atp8a1 at the early developmental stage of postnatal day 6 (P6). Transmission electron microscopy (TEM) analysis of the lentivirus-Atp8a1 treated (Atp8a1 +) mice in adulthood revealed fewer and weaker excitatory synapses in the hippocampal CA1 region compared to mice injected with empty virus. Significant inhibition of the Schaffer collateral pathway was observed in the Atp8a1 + mice in paired-pulse recording (PPR) at 20-ms inter-stimulus interval. In the three-chambered sociability test, the Atp8a1 + mice displayed no preference for an encaged stranger mouse over a novel object, which is a characteristic autistic-like behavior. In sharp contrast, Atp8a1 (/) mice displayed a preference for a stranger mouse over the novel object, which is characteristic of neurotypical mouse behavior. However, similar to the Atp8a1 + mice, the Atp8a1 (/) mice harbored fewer and weaker excitatory synapses in CA1 compared to wild-type controls, and displayed inhibition at 20-ms inter-stimulus interval in PPR. These findings suggest that both elevated and diminished levels of Atp8a1 during early development are detrimental to brain connectivity, but only elevated Atp8a1 is associated with aberrant social behavior. Mice with augmented levels of Atp8a1 may therefore serve as a potential model in autism research.
Keywords: Autism; Atp8a1; Flippase; Synaptic strength; TEM; Lenti-virus;

Contribution of polymorphic variation of inositol hexakisphosphate kinase 3 (IP6K3) gene promoter to the susceptibility to late onset Alzheimer's disease by Paolina Crocco; Adolfo Saiardi; Miranda S. Wilson; Raffaele Maletta; Amalia C. Bruni; Giuseppe Passarino; Giuseppina Rose (1766-1773).
Maintenance of electric potential and synaptic transmission are energetically demanding tasks that neuronal metabolism must continually satisfy. Inability to fulfil these energy requirements leads to the development of neurodegenerative disorders, including Alzheimer's disease. A prominent feature of Alzheimer's disease is in fact neuronal glucose hypometabolism. Thus understanding the fine control of energetic metabolism might help to understand neurodegenerative disorders. Recent research has indicated that a novel class of signalling molecules, the inositol pyrophosphates, act as energy sensors. They are able to alter the balance between mitochondrial oxidative phosphorylation and glycolytic flux, ultimately affecting the cellular level of ATP. The neuronal inositol pyrophosphate synthesis relies on the activity of the neuron enriched inositol hexakisphosphate kinase 3 (IP6K3) enzyme. To verify an involvement of inositol pyrophosphate signalling in neurodegenerative disorders, we performed tagging single nucleotide polymorphism (SNP) analysis of the IP6K3 gene in patients with familial and sporadic late onset Alzheimer's disease (LOAD). Two SNPs in the 5′-flanking promoter region of the IP6K3 gene were found to be associated with sporadic LOAD. Characterizing the functionality of the two polymorphisms by luciferase assay revealed that one of them (rs28607030) affects IP6K3 promoter activity, with the G allele showing an increased activity. As the same allele has a beneficial effect on disease risk, this may be related to upregulation of IP6K3 expression, with a consequent increase in inositol pyrophosphate synthesis. In conclusion, we provide the first evidence for a contribution of genetic variability in the IP6K3 gene to LOAD pathogenesis.
Keywords: IP6K3; Inositol pyrophosphate; IP7; Metabolism; SNP; Alzheimer's disease;

Nuclear receptor NR5A2 is involved in the calreticulin gene regulation during renal fibrosis by Eleni Arvaniti; Athina Vakrakou; Valeria Kaltezioti; Athanasios Stergiopoulos; Niki Prakoura; Panagiotis K. Politis; Aristidis Charonis (1774-1785).
Renal fibrosis is a common histological finding present in many pathologies; however, key signaling pathways and molecular determinants involved in the development of fibrosis are not fully known yet. Previous findings have established a causative role of calreticulin's up-regulation during the development of renal fibrosis while its down-regulation exhibited a protective effect against fibrosis. Therefore, the mechanism of its up-regulation needs to be explored.Bioinformatics analyses of the calreticulin gene promoter combined with transcriptional assays and in vivo chromatin immunoprecipitation experiments in the Unilateral Ureteric Obstruction (UUO) model of renal fibrosis, indicated that NR5A2 is a critical regulator of calreticulin expression. To confirm this finding, and further study post-translational modifications of NR5A2, real time RT-qPCR, immunohistochemistry and Western blotting experiments were performed.NR5A2 is up-regulated at both mRNA and protein level during kidney fibrosis in the UUO model. The post-translational modification of SUMOylation was identified as a critical parameter in this phenomenon and SUMOylation was observed to be up-regulated during the development of renal fibrosis. The enzyme Ubc9, critical for the process of SUMOylation was also upregulated at mRNA and protein level during the process.These data establish for the first time a role for NR5A2 and its SUMOylation on the transcriptional regulation of the calreticulin gene in a rodent model of renal fibrosis and raise the possibility that NR5A2 might be a novel target for future anti-fibrotic interventions.
Keywords: Fibrosis; Obstructive nephropathy; Proximal tubule; Transcription regulation;

Alzheimer's disease (AD) is currently one of the most common neurodegenerative disorders worldwide. To date, no cure has been developed for AD, and some disease-modifying treatments show side effects and low efficacy. Increasing evidence shows that cyanidin 3-O-β-glucopyranoside (Cy3G), which is naturally derived from many plants, may provide protection against neurodegenerative diseases including AD; however, its exact role is still unclear. Therefore, we investigated the mechanisms of the effects of Cy3G on beta-amyloid 25–35 (Aβ25–35)-induced SH-SY5Y cell injury and cognitive impairment in the APPswe/PS1ΔE9 (PAP) mouse model of AD. Furthermore, we aimed to determine the molecular target initiated by Cy3G. The data indicated that Cy3G-mediated neuroprotection involved the inhibition of Aβ25–35 binding to the cell surface and spontaneous aggregation of Aβ25–35 fibrils at the molecular level. Furthermore, in an in vitro study, Aβ25–35-mediated cytotoxicity, which was caused by inducing apoptotic cell death and ROS formation, was also ameliorated by Cy3G intervention. In addition, upregulation of peroxisome proliferator-activated receptor-γ (PPARγ) protein involved in glucose/lipid metabolism by Cy3G treatment verified that the initiated molecule was Cy3G. In an in vivo study, Cy3G was shown to alleviate cognitive impairment, improve cerebral glucose uptake and decrease fasting blood glucose levels. In conclusion, Cy3G ameliorates amyloid β peptide-induced injury both in vitro and in vivo through the PPARγ pathway. Thus, Cy3G has a good safety profile as a potential natural PPARγ agonist and may be used as an ideal alternative to traditional disease-modifying treatments against AD.
Keywords: Alzheimer's disease; Cyanidin 3-O-β-glucopyranoside; Aβ25–35; APPswe/PS1ΔE9 mice; PPARγ; Neuroprotection;

Overexpression of the short endoglin isoform reduces renal fibrosis and inflammation after unilateral ureteral obstruction by José M. Muñoz-Félix; Lucía Pérez-Roque; Elena Núñez-Gómez; Bárbara Oujo; Miguel Arévalo; Laura Ruiz-Remolina; Cristina Cuesta; Carmen Langa; Fernando Pérez-Barriocanal; Carmelo Bernabeu; José M. Lopez-Novoa (1801-1814).
Transforming growth factor beta 1 (TGF-β1) is one of the most studied cytokines involved in renal tubulo-interstitial fibrosis, which is characterized by myofibroblast abundance and proliferation, and high buildup of extracellular matrix in the tubular interstitium leading to organ failure. Endoglin (Eng) is a 180-kDa homodimeric transmembrane protein that regulates a great number of TGF-β1 actions in different biological processes, including ECM synthesis. High levels of Eng have been observed in experimental models of renal fibrosis or in biopsies from patients with chronic kidney disease. In humans and mice, two Eng isoforms are generated by alternative splicing, L-Eng and S-Eng that differ in the length and composition of their cytoplasmic domains. We have previously described that L-Eng overexpression promotes renal fibrosis after unilateral ureteral obstruction (UUO). However, the role of S-Eng in renal fibrosis is unknown and its study would let us analyze the possible function of the cytoplasmic domain of Eng in this process. For this purpose, we have generated a mice strain that overexpresses S-Eng (S-ENG+) and we have performed an UUO in S-ENG+ and their wild type (WT) control mice. Our results indicate that obstructed kidney of S-ENG+ mice shows lower levels of tubulo-interstitial fibrosis, less inflammation and less interstitial cell proliferation than WT littermates. Moreover, S-ENG+ mice show less activation of Smad1 and Smad2/3 pathways. Thus, S-Eng overexpression reduces UUO-induced renal fibrosis and some associated mechanisms. As L-Eng overexpression provokes renal fibrosis we conclude that Eng-mediated induction of renal fibrosis in this model is dependent on its cytoplasmic domain.
Keywords: Fibrosis; Endoglin; TGF-β; Kidney; Animal model; Obstructive nephropathy;

MDMA is one of the most used drugs by adolescents and its consumption has been associated with many psychobiological problems, among them psychomotor problems. Moreover, some authors described that early exposure to MDMA may render the dopaminergic neurons more vulnerable to the effects of future neurotoxic insults. Alzheimer disease (AD) is the main cause of dementia in the elderly and a percentage of the patients have predisposition to suffer nigrostriatal alterations, developing extrapyramidal signs. Nigrostriatal dysfunction in the brain of aged APPswe/PS1dE9 (APP/PS1), a mouse model of familiar AD (FAD), has also been described. The aim of the present study was to investigate the consequences of adolescent exposure to MDMA in APP/PS1 mice, on nigrostriatal function on early adulthood. We used a MDMA schedule simulating weekend binge abuse of this substance. Our MDMA schedule produced a genotype-independent decrease in dopaminergic neurons in the substantia nigra that remained at least 3 months. Shortly after the injury, wild-type animals showed a decrease in the locomotor activity and apparent DA depletion in striatum, however in the APP/PS1 mice neither the locomotor activity nor the DA levels were modified, but a reduction in dopamine transporter (DAT) expression and a higher levels of oxidative stress were observed. We found that these disturbances are age-related characteristics that this APP/PS1 mice develops spontaneously much later. Therefore, MDMA administration seems to anticipate the striatal dopaminergic dysfunction in this FAD model. The most important outcome lies in a potentiation, by MDMA, of the amyloid beta deposition in the striatum.
Keywords: 3.4-Methylenedioxymethamphetamine; APPswe/PS1dE9 mice; Striatum; Dopaminergic dysfunction; Amyloid plaques;

Signaling mechanisms underlying the glioprotective effects of resveratrol against mitochondrial dysfunction by Bruna Bellaver; Larissa Daniele Bobermin; Débora Guerini Souza; Marília Danielly Nunes Rodrigues; Adriano Martimbianco de Assis; Moacir Wajner; Carlos-Alberto Gonçalves; Diogo Onofre Souza; André Quincozes-Santos (1827-1838).
Resveratrol, a polyphenol found in grapes and red wine, exhibits antioxidant, anti-inflammatory, anti-aging and, neuroprotective effects. Resveratrol also plays a significant role modulating glial functionality, protecting the health of neuroglial cells against several neuropsychiatric in vivo and in vitro experimental models. Mitochondrial impairment strongly affected astrocyte functions and consequently brain homeostasis. Molecules that promote astrocyte mitochondrial protection are fundamental to maintain brain energy balance and cellular redox state, contributing to brain healthy. Thus, the present study was designed to evaluate some glioprotective mechanisms of resveratrol against mitochondrial damage promoted by azide exposure in hippocampal primary astrocyte cultures. Azide treatment provoked deleterious effects, including the dysfunction of mitochondria, the deterioration of redox homeostasis, the augmentation of pro-inflammatory cytokines and impairment of glutamate uptake activity. However, resveratrol prevented these effects, protecting hippocampal astrocytes against azide-induced cytotoxicity through the heme-oxygenase-1 (HO-1) pathway and inhibiting p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NFκB) activation. Resveratrol also protected astrocytes via phosphatidylinositide 3-kinase (PI3K)/Akt. These results contribute to the comprehension of the mechanisms by which resveratrol mediates hippocampal astrocyte protection against mitochondrial failure and implicate resveratrol as an important glioprotective molecule.
Keywords: Resveratrol; Mitochondrial dysfunction; Heme oxygenase-1 (HO-1); Hippocampal astrocytes; Nuclear factor kappa B (NFκB);

Cerulein-induced pancreatic fibrosis is modulated by Smad7, the major negative regulator of transforming growth factor-β signaling by Xuan Li; Salvatore Nania; Nino Fejzibegovic; Carlos Fernández Moro; Lena Klopp-Schulze; Caroline Verbeke; J.-Matthias Löhr; Rainer L. Heuchel (1839-1846).
Chronic pancreatitis is the most common disease of the exocrine pancreas, characterized by progressive inflammation, acinar atrophy and fibrosis. Transforming growth factor-β signaling (TGFβ) is the most potent fibrogenic cytokine known, and its increased expression is a common denominator for fibrosis in chronic pancreatitis. Smad7 is induced by the TGFβ superfamily members as an intracellular inhibitory feedback antagonizing TGFβ signaling. To investigate the functional role of Smad7 in vivo, we induced chronic pancreatitis by repeated administration of cerulein in mice that are deficient in exon-I of Smad7. The response to chronic pancreatitis induction was significantly more severe in Smad7 mutant mice as indicated by a stronger accumulation of extracellular matrix, increased levels of inflammatory cells and an elevated number of mesenchymal cells/myofibroblasts in Smad7 mutant pancreata. Taken together, we conclude that lack of a functional Smad7 gene results in more severe damage in chronic pancreatitis. Therefore, Smad7 could be envisaged as a promising target in antifibrotic therapy of the pancreas.
Keywords: Smad7; TGFβ; Chronic pancreatitis; Fibrosis;