BBA - Molecular Basis of Disease (v.1802, #11)
Editorial Board (i).
New insights into the role of mitochondrial dysfunction and protein aggregation in Parkinson's disease by Weilin Xie; Oi Wan Wan; Kenny K.K. Chung (935-941).
Parkinson's disease (PD) is a common neurodegenerative movement disorder that affects increasing number of elderly in the world population. The disease is caused by a selective degeneration of dopaminergic neurons in the substantia nigra pars compacta with the molecular mechanism underlying this neurodegeneration still not fully understood. However, various studies have shown that mitochondrial dysfunction and abnormal protein aggregation are two of the major contributors for PD. In fact this notion has been supported by recent studies on genes that are linked to familial PD (FPD). For instance, FPD linked gene products such as PINK1 and parkin have been shown to play critical roles in the quality control of mitochondria, whereas α-synuclein has been found to be the major protein aggregates accumulated in PD patients. These findings suggest that further understanding of how dysfunction of these pathways in PD will help develop new approaches for the treatment of this neurodegenerative disorder.►Oxidative stress and mitochondrial dysfunction in Parkinson's disease. ►PINK1 and parkin in the quality control of mitochondria. ►Protein aggregation, the ubiquitin proteasomal system and the autophagy lysosomal pathway in Parkinson's disease.
Keywords: Neurodegeneration; PINK1; Parkin; Ubiquitin proteasomal system; Autophagy lysosomal pathway;
Cortical and hippocampal mitochondria bioenergetics and oxidative status during hyperglycemia and/or insulin-induced hypoglycemia by Susana Cardoso; Maria S. Santos; Raquel Seiça; Paula I. Moreira (942-951).
This study was undertaken to evaluate the effects of streptozotocin (STZ)-induced hyperglycemia and insulin-induced hypoglycemia in cortical and hippocampal mitochondria bioenergetics and oxidative status. For that purpose we used, citrate (vehicle)-treated Wistar rats, STZ-treated rats [i.p., 50 mg/kg body weight] and STZ-treated rats injected with insulin [s.c., dose adjusted to blood glucose levels] 1 h prior to sacrifice to induce an acute episode of hypoglycemia. Several parameters were analyzed: respiratory chain, phosphorylation system, thiobarbituric acid reactive substances (TBARS) levels, hydrogen peroxide (H2O2) production rate, and non-enzymatic and enzymatic antioxidant defenses. Cortical mitochondria from insulin-induced hypoglycemic rats present a significant decrease in the ADP/O index, a significant increase in the repolarization lag phase and a decrease in GSH/GSSG ratio when compared with STZ and control mitochondria. Both STZ-induced diabetes and insulin-induced hypoglycemia promote a significant increase in TBARS levels and a decrease in glutathione disulfide reductase activity. Diabetic cortical mitochondria present a significant decrease in glutathione peroxidase (GPx) activity compared to control mitochondria. In turn, insulin-induced hypoglycemia induced a significant increase in GPx and manganese superoxide dismutase (MnSOD) activities. In hippocampal mitochondria, insulin-induced hypoglycemia increases the respiratory control ratio whereas both situations, hyper- and hypoglycemia, potentiate H2O2 production and decrease the activity of MnSOD. These results suggest that the poor glycemic control that occurs in type 1 diabetic patients undergoing insulin therapy may have detrimental effects in brain areas involved in learning and memory.
Keywords: Cortex; Hippocampus; Hyperglycemia; Insulin-induced hypoglycemia; Mitochondria; Oxidative stress;
Magnesium deficiency promotes a pro-atherogenic phenotype in cultured human endothelial cells via activation of NFkB by Silvia Ferrè; Erika Baldoli; Marzia Leidi; Jeanette A.M. Maier (952-958).
Phenotypic modulation of endothelium to a dysfunctional state contributes to the pathogenesis of atherosclerosis, partly through the activation of the transcription factor NFkB. Several data indicate that magnesium deficiency caused by prolonged insufficient intake and/or defects in its homeostasis may be a missing link between diverse cardiovascular risk factors and atherosclerosis. Here we report that endothelial cells cultured in low magnesium rapidly activate NFkB, an event which is prevented by exposure to the anti-oxidant trolox. It is well known that NFkB activation correlates with marked alterations of the cytokine network. In the present study, we show that exposure of endothelial cells to low magnesium increases the secretion of RANTES, interleukin 8 and platelet derived growth factor BB, all important players in atherogenesis. Moreover, we describe the increased secretion of matrix metalloprotease-2 and -9 and of their inhibitor TIMP-2. Interestingly, by zymography we show that metalloprotease activity predominated over the inhibitory effect of TIMP-2.These results indicate that low magnesium promotes endothelial dysfunction by inducing pro-inflammatory and pro-atherogenic events.
Keywords: Endothelial cell; Magnesium; NFkB; Cytokine; MMP;
Different altered pattern expression of genes related to apoptosis in isolated methylmalonic aciduria cblB type and combined with homocystinuria cblC type by Ana Jorge-Finnigan; Alejandra Gámez; Belén Pérez; Magdalena Ugarte; Eva Richard (959-967).
An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders involved in cobalamin metabolism, including isolated methylmalonic aciduria (MMA) cblB type and MMA combined with homocystinuria (MMAHC) cblC type. Given the relevance of p38 and JNK kinases in stress-response, their activation in fibroblasts from a spectrum of patients (mut, cblA, cblB, cblC and cblE) was analyzed revealing an increased expression of the phosphorylated-forms, specially in cblB and cblC cell lines that presented the highest ROS and apoptosis levels. To gain further insight into the molecular mechanisms responsible for the enhanced apoptotic process observed in cblB and cblC fibroblasts, we evaluated the expression pattern of 84 apoptosis-related genes by quantitative real-time PCR. An elevated number of pro-apoptotic genes were overexpressed in cblC cells showing a higher rate of apoptosis compared to cblB and control samples. Additionally, apoptosis appears to be mainly triggered through the extrinsic pathway in cblC, while the intrinsic pathway was primarily activated in cblB cells. The differences observed regarding the apoptosis rate and preferred pathway between cblB and cblC patients, who both built up methylmalonic acid, might be explained by the accumulated homocysteine in the cblC group. The loss of MMACHC function in cblC patients might be partially responsible for the oxidative stress and apoptosis processes observed in these cell lines. Our results suggest that ROS production may represent a genetic modifier of the phenotype and support the potential of using antioxidants as a novel therapeutic strategy to improve the severe neurological outcome of these rare diseases.►p38 and JNK are activated in patients' cell lines with defects in cobalamin metabolism. ►CblB and cblC cell lines do not share a common apoptotic pathway. ►Homocysteine could be responsible for the differences between cblB and cblC groups. ►MMACHC loss of function in cblC patients might be partially responsible for oxidative stress and apoptosis. ►Antioxidants could represent a novel therapeutic strategy in MMAHC.
Keywords: Methylmalonic aciduria; Homocystinuria; ROS; Apoptosis; PCR array; Stress-kinase;
Biological effects of mutant ceruloplasmin on hepcidin-mediated internalization of ferroportin by Satoshi Kono; Kenichi Yoshida; Naohisa Tomosugi; Tatsuhiro Terada; Yasushi Hamaya; Shigeru Kanaoka; Hiroaki Miyajima (968-975).
Ceruloplasmin plays an essential role in cellular iron efflux by oxidizing ferrous iron exported from ferroportin. Ferroportin is posttranslationally regulated through internalization triggered by hepcidin binding. Aceruloplasminemia is an autosomal recessive disorder of iron homeostasis resulting from mutations in the ceruloplasmin gene. The present study investigated the biological effects of glycosylphosphatidylinositol (GPI)-linked ceruloplasmin on the hepcidin-mediated internalization of ferroportin. The prevention of hepcidin-mediated ferroportin internalization was observed in the glioma cells lines expressing endogenous ceruloplasmin as well as in the cells transfected with GPI-linked ceruloplasmin under low levels of hepcidin. A decrease in the extracellular ferrous iron by an iron chelator and incubation with purified ceruloplasmin in the culture medium prevented hepcidin-mediated ferroportin internalization, while the reconstitution of apo-ceruloplasmin was not able to prevent ferroportin internalization. The effect of ceruloplasmin on the ferroportin stability was impaired due to three distinct properties of the mutant ceruloplasmin: namely, a decreased ferroxidase activity, the mislocalization in the endoplasmic reticulum, and the failure of copper incorporation into apo-ceruloplasmin. Patients with aceruloplasminemia exhibited low serum hepcidin levels and a decreased ferroportin protein expression in the liver. The in vivo findings supported the notion that under low levels of hepcidin, mutant ceruloplasmin cannot stabilize ferroportin because of a loss-of-function in the ferroxidase activity, which has been reported to play an important role in the stability of ferroportin. The properties of mutant ceruloplasmin regarding the regulation of ferroportin may therefore provide a therapeutic strategy for aceruloplasminemia patients.►Hepcidin and ceruloplasmin were competitors affecting ferroportin internalization. ►Ceruloplasmin stabilized ferroportin under low hepcidin conditions. ►Ferroxidase activity played an important role in the stability of ferroportin. ►Mutant ceruloplasmin with low ferroxidase activity impaired ferroportin stability. ►Serum hepcidin levels and hepatic ferroportin were decreased in aceruloplasminemia.
Keywords: Aceruloplasminemia; Ceruloplasmin; Ferroportin; Haemochromatosis; Hepcidin;
Metabolic regulation of APOBEC-1 Complementation Factor trafficking in mouse models of obesity and its positive correlation with the expression of ApoB protein in hepatocytes by Chad A. Galloway; John Ashton; Janet D. Sparks; Robert A. Mooney; Harold C. Smith (976-985).
APOBEC-1 Complementation Factor (ACF) is an RNA-binding protein that interacts with apoB mRNA to support RNA editing. ACF traffics between the cytoplasm and nucleus. It is retained in the nucleus in response to elevated serum insulin levels where it supports enhanced apoB mRNA editing. In this report we tested whether ACF may have the ability to regulate nuclear export of apoB mRNA to the sites of translation in the cytoplasm. Using mouse models of obesity-induced insulin resistance and primary hepatocyte cultures we demonstrated that both nuclear retention of ACF and apoB mRNA editing were reduced in the livers of hyperinsulinemic obese mice relative to lean controls. Coincident with an increase in the recovery of ACF in the cytoplasm was an increase in the proportion of total cellular apoB mRNA recovered in cytoplasmic extracts. Cytoplasmic ACF from both lean controls and obese mouse livers was enriched in endosomal fractions associated with apoB mRNA translation and ApoB lipoprotein assembly. Inhibition of ACF export to the cytoplasm resulted in nuclear retention of apoB mRNA and reduced both intracellular and secreted ApoB protein in primary hepatocytes. The importance of ACF for modulating ApoB was supported by the finding that RNAi knockdown of ACF reduced ApoB secretion. An additional discovery from this study was the finding that leptin is a suppressor ACF expression. Dyslipidemia is a common pathology associated with insulin resistance that is in part due to the loss of insulin controlled secretion of lipid in ApoB-containing very low density lipoproteins. The data from animal models suggested that loss of insulin regulated ACF trafficking and leptin regulated ACF expression may make an early contribution to the overall pathology associated with very low density lipoprotein secretion from the liver in obese individuals.
Keywords: Diabetes; Insulin resistance; APOBEC-1 Complementation Factor; Apolipoprotein B; mRNA editing;
Impaired phospholipases A2 production by stimulated macrophages from patients with acute respiratory distress syndrome by Eleana Hatzidaki; George Nakos; Eftychia Galiatsou; Marilena E. Lekka (986-994).
The aim of this study was to investigate whether early phase of acute respiratory distress syndrome (ARDS) is associated with changes in immune response, either systemic or localized to the lung. ARDS and control mechanically ventilated patients, as well as healthy volunteers were studied. Alveolar macrophages (AMΦ) and blood monocytes (BM) were treated ex vivo with lipopolysaccharide (LPS), interferon-γ (IFNγ), and surfactant. Phospholipase A2 (PLA2) activity and TLR4 expression were evaluated as markers of cell response. AMΦ from ARDS patients did not respond upon treatment with either LPS or IFN-γ by inducing PLA2 production. On the contrary, upon stimulation, in control patients the intracellular PLA2, (mainly cPLA2) levels were increased, but secretion of PLA2 (mainly sPLA2-IIA) was observed only after treatment with LPS. Surfactant suppressed PLA2 production in cells from both groups of patients. Increased relative changes of total PLA2 activity and an upregulation of TLR4 expression upon stimulation was observed in BM from primary ARDS, control patients and healthy volunteers. In BM from secondary ARDS patients, however, no PLA2 induction was observed, with a concomitant down-regulation of TLR4 expression. Cytosolic PLA2, its activated form, p-cPLA2, and sPLA2-IIA were the predominant PLA2 types within the cells, while extracellularly only sPLA2-IIA was identified. These results support the concept of down-regulated innate immunity in early ARDS that is compartmentalized in primary and systemic in secondary ARDS. PLA2 isoforms could serve as markers of the immunity status in ARDS. Finally, our data highlight the role of surfactant in controlling inflammation.
Keywords: ARDS; Phospholipase A2; Alveolar macrophage; Lung surfactant; Immune tolerance;
Untranslated regions of thyroid hormone receptor beta 1 mRNA are impaired in human clear cell renal cell carcinoma by Adam Master; Anna Wójcicka; Agnieszka Piekiełko-Witkowska; Joanna Bogusławska; Piotr Popławski; Zbigniew Tański; Veerle M. Darras; Graham R. Williams; Alicja Nauman (995-1005).
Thyroid hormone receptor β1 (TRβ1) is a hormone-dependent transcription factor activated by 3,5,3'-l-triiodothyronine (T3). TRβ1 functions as a tumor suppressor and disturbances of the THRB gene are frequent findings in cancer. Translational control mediated by untranslated regions (UTRs) regulates cell proliferation, metabolism and responses to cellular stress, processes that are involved in carcinogenesis. We hypothesized that reduced TRβ1 expression in clear cell renal cell cancer (ccRCC) results from regulatory effects of TRβ1 5′ and 3′UTRs on protein translation. We determined TRβ1 expression and alternative splicing of TRβ1 5′ and 3′UTRs in ccRCC and control tissue together with expression of the type 1 deiodinase enzyme (coded by DIO1, a TRβ1 target gene). Tissue concentrations of T3 (which are generated in part by D1) and expression of miRNA-204 (an mRNA inhibitor for which a putative interaction site was identified in the TRβ1 3′UTR) were also determined. TRβ1 mRNA and protein levels were reduced by 70% and 91% in ccRCC and accompanied by absent D1 protein, a 58% reduction in tissue T3 concentration and 2-fold increase in miRNA-204. Structural analysis of TRβ1 UTR variants indicated that reduced TRβ1 expression may be maintained in ccRCC by posttranscriptional mechanisms involving 5′UTRs and miRNA-204. The tumor suppressor activity of TRβ1 indicates that reduced TRβ1 expression and tissue hypothyroidism in ccRCC tumors is likely to be involved in the process of carcinogenesis or in maintaining a proliferative advantage to malignant cells.►TRβ1 mRNA and protein levels are significantly reduced in renal cancer. ►Loss of D1 and reduced level of intracellular T3 are found in kidney tumors. ►TRβ1 decrease in renal cancer is caused by miRNA-204 upregulation. ►5′UTR and 3′UTR are involved in posttranscriptional TRβ1 regulation in renal cancer. ►Renal cancer is characterized by tissue hypothyroidism.
Keywords: Translational control in cancer; Untranslated region; miRNA; Thyroid hormone receptor beta 1; Thyroid hormone; Renal cancer; ccRCC;
Evidence of reactive astrocytes but not peripheral immune system activation in a mouse model of Fragile X syndrome by Christopher J. Yuskaitis; Eleonore Beurel; Richard S. Jope (1006-1012).
Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is one of the few known genetic causes of autism. FXS results from the loss of Fmr1 gene function; thus, Fmr1 knockout mice provide a model to study impairments associated with FXS and autism and to test potential therapeutic interventions. The inhibitory serine phosphorylation of glycogen synthase kinase-3 (GSK3) is lower in brain regions of Fmr1 knockout mice than wild-type mice and the GSK3 inhibitor lithium rescues several behavioral impairments in Fmr1 knockout mice. Therefore, we examined if the serine phosphorylation of GSK3 in Fmr1 knockout mice also was altered outside the brain and if administration of lithium ameliorated the macroorchidism phenotype. Additionally, since GSK3 regulates numerous functions of the immune system and immune alterations have been associated with autism, we tested if immune function is altered in Fmr1 knockout mice. The inhibitory serine phosphorylation of GSK3 was significantly lower in the testis and liver of Fmr1 knockout mice than wild-type mice, and chronic lithium treatment reduced macroorchidism in Fmr1 knockout mice. No alterations in peripheral immune function were identified in Fmr1 knockout mice. However, examination of glia, the immune cells of the brain, revealed reactive astrocytes in several brain regions of Fmr1 knockout mice and treatment with lithium reduced this in the striatum and cerebellum. These results provide further evidence of the involvement of dysregulated GSK3 in FXS, and demonstrate that lithium administration reduces macroorchidism and reactive astrocytes in Fmr1 knockout mice.
Keywords: Astrocytes; Fragile X syndrome; Glycogen synthase kinase-3; Lithium; Macroorchidism;
Oxidized low density lipoprotein inhibits phosphate signaling and phosphate-induced mineralization in osteoblasts. Involvement of oxidative stress by Cécile Mazière; Valéry Savitsky; Antoine Galmiche; Cathy Gomila; Ziad Massy; Jean-Claude Mazière (1013-1019).
It is well admitted that oxidized LDL (OxLDL) plays a major role in the generation and progression of atherosclerosis. Since atherosclerosis is often accompanied by osteoporosis, the effects of OxLDL on phosphate-induced osteoblast mineralization were investigated.Calcium deposition, expression of osteoblast markers and inorganic phosphate (Pi) signaling were determined under OxLDL treatment.OxLDL, within the range of 10–50 μg protein/ml, inhibited Pi-induced UMR106 rat osteoblast mineralization. In parallel, the expression of Cbfa1/Runx2 transcription factor was decreased, and the intracellular level of the osteoblast marker osteopontin (OPN) was reduced. The extracellular level of another marker, receptor activator of nuclear factor kappa B ligand (RANKL), was also diminished. OxLDL inhibited Pi signaling via ERK/JNK kinases and AP1/CREB transcription factors. OxLDL triggered the generation of reactive oxygen species (ROS), either in the absence or presence of Pi. Furthermore, the effects of OxLDL on Pi-induced mineralization, generation of ROS and extracellular level OPN were reproduced by the lipid extract of the particle, whereas the antioxidant vitamin E prevented them.This work demonstrates that OxLDL, by generation of an oxidative stress, inhibits of Pi signaling and impairs Pi-induced osteoblast differentiation.This highlights the role of OxLDL in bone remodeling and in degenerative disorders other than atherosclerosis, especially in osteoporosis.►This work demonstrates that OxLDL, by generation of an oxidative stress, inhibits of Pi signaling and impairs Pi-induced osteoblast differentiation. This highlights the role of OxLDL in bone remodeling and in degenerative disorders other than atherosclerosis, especially in osteoporosis.
Keywords: Osteoblasts; Differentiation; OxLDL; Oxidative stress; Signaling;
Poly(ADP-ribose) polymerase-1 (PARP-1) gene deficiency alleviates diabetic kidney disease by Hanna Shevalye; Yury Maksimchyk; Pierre Watcho; Irina G. Obrosova (1020-1027).
Poly(ADP-ribose)polymerase (PARP) inhibitors prevent or alleviate diabetic nephropathy. This study evaluated the role for PARP-1 in diabetic kidney disease using the PARP-1-deficient mouse. PARP-1−/− and the wild-type (129S1/SvImJ) mice were made diabetic with streptozotocin, and were maintained for 12 weeks. Final blood glucose concentrations were increased ∼ 3.7-fold in both diabetic groups. PARP-1 protein expression (Western blot analysis) in the renal cortex was similar in non-diabetic and diabetic wild-type mice (100% and 107%) whereas all knockouts were PARP-1-negative. PARP-1 gene deficiency reduced urinary albumin (ELISA) and protein excretion prevented diabetes-induced kidney hypertrophy, and decreased mesangial expansion and collagen deposition (both assessed by histochemistry) as well as fibronectin expression. Renal podocyte loss (immunohistochemistry) and nitrotyrosine and transforming growth factor-β1 accumulations (both by ELISA) were slightly lower in diabetic PARP-1−/− mice, but the differences with diabetic wild-type group did not achieve statistical significance. In conclusion, PARP-1−/− gene deficiency alleviates although does not completely prevent diabetic kidney disease.► PARP-1 activation is responsible for kidney hypertrophy and contributes to albuminuria, proteinuria, renal mesangial expansion, fibronectin overexpression, and collagen deposition in Type 1 diabetic kidney disease. ► The nephroprotective effect of PARP-1 gene deficiency was less pronouced than that of non-selective PARP inhibitors in previous studies which suggests that the PARP isoform other than PARP-1 are also involved in the pathogenesis of diabetic nephropathy. ► The findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies, for prevention and treatment of diabetic kidney disease.
Keywords: Albuminuria; Collagen; Diabetic nephropathy; Mesangial expansion; Poly(ADP-ribose) polymerase-1; Poly(ADP-ribose)polymerase-1 deficient mouse; Renal podocyte;
Mitochondrial involvement and erythronic acid as a novel biomarker in transaldolase deficiency by Udo F.H. Engelke; Fokje S.M. Zijlstra; Fanny Mochel; Vassili Valayannopoulos; Daniel Rabier; Leo A.J. Kluijtmans; András Perl; Nanda M. Verhoeven-Duif; Pascale de Lonlay; Mirjam M.C. Wamelink; Cornelis Jakobs; Éva Morava; Ron A. Wevers (1028-1035).
Sedoheptulose, arabitol, ribitol, and erythritol have been identified as key diagnostic metabolites in TALDO deficiency.Urine from 6 TALDO-deficient patients and TALDO-deficient knock-out mice were analyzed using 1H-NMR spectroscopy and GC–mass spectrometry.Our data confirm the known metabolic characteristics in TALDO-deficient patients. The β-furanose form was the major sedoheptulose anomer in TALDO-deficient patients. Erythronic acid was identified as a major abnormal metabolite in all patients and in knock-out TALDO mice implicating an as yet unknown biochemical pathway in this disease. A putative sequence of enzymatic reactions leading to the formation of erythronic acid is presented. The urinary concentration of the citric acid cycle intermediates 2-oxoglutaric acid and fumaric acid was increased in the majority of TALDO-deficient patients but not in the knock-out mice.Erythronic acid is a novel and major hallmark in TALDO deficiency. The pathway leading to its production may play a role in healthy humans as well. In TALDO-deficient patients, there is an increased flux through this pathway. The finding of increased citric acid cycle intermediates hints toward a disturbed mitochondrial metabolism in TALDO deficiency.► Urine 1H-NMR Spectroscopy can be used to diagnose patients with TALDO-deficiency ► Erythronic acid is a novel and major hallmark in TALDO-deficiency ► Increased citric acid cycle intermediates hints towards a disturbed mitochondrial metabolism in TALDO-deficiency.
Keywords: NMR spectroscopy; Citric acid cycle intermediates; Pentose phosphate pathway; Transaldolase deficiency; Polyols; Sedoheptulose; Erythronic acid; 2-Oxoglutaric acid;
Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis by Sergio Perez-Alvarez; Maria D. Cuenca-Lopez; Raquel M. Melero-Fernández de Mera; Elena Puerta; Andonis Karachitos; Piotr Bednarczyk; Hanna Kmita; Norberto Aguirre; Maria F Galindo; Joaquin Jordán (1036-1047).
Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-XL and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a “bioenergetic crisis.” A decreased level of cellular energy may predispose cells to necrotic-like cell death.►In SH-SY5Y cells high concentrations of methadone were required to induce cell death. ►Methadone-induced cell death seems to be related to necrotic processes rather than apoptosis. ►It involves Bax translocation to the mitochondria, accompanied with cytochrome c release. ►Methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. ►Methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles.
Keywords: Methadone; Mitochondria; Necrosis; Neurodegeneration; Bax; Clark electrode;
Hyperthermic preconditioning protects astrocytes from ischemia/reperfusion injury by up-regulation of HIF-1 alpha expression and binding activity by Fang Du; Li Zhu; Zhong-Ming Qian; Xiao-Mei Wu; Wing-Ho Yung; Ya Ke (1048-1053).
It has been demonstrated that hypoxia-inducible factor-1 alpha (HIF-1 alpha) mediates ischemic tolerance induced by hypoxia/ischemia or pharmacological preconditioning. In addition, preconditioning stimuli can be cross-tolerant, safeguarding against other types of injury. We therefore hypothesized HIF-1 alpha might also be associated with ischemic tolerance induced by hyperthermic preconditioning. In the present study, we demonstrated for the first time that 6 h of hyperthermia (38 °C or 40 °C) could induce a characteristic “reactive” morphology and a significant increase in the expression of bystin in astrocytes. We also showed that pre-treatment with 6 h of hyperthermia resulted in a significant increase in cell viability and a remarkable decrease in lactate dehydrogenase (LDH) release and apoptosis development in the astrocytes that were exposed to 24 h of ischemia and a subsequent 24 h of reperfusion. Analysis of mechanisms showed that hyperthermia could lead to a significant increase in HIF-1 alpha expression and also the HIF-1 binding activity in the ischemia/reperfusion astrocytes. The data provide evidence to our hypothesis that the up-regulation of HIF-1 alpha is associated with the protective effects of hyperthermic preconditioning on astrocytes against ischemia/reperfusion injury.
Keywords: Hyperthermiac preconditioning; Ischemia/reperfusion; Reactive astrocytes; Hypoxia-inducible factor-1 alpha (HIF-1 alpha); Apoptosis; Cell viability; Lactate dehydrogenase (LDH);
Role of IL-6 trans-signaling in CCl4 induced liver damage by Jessica Gewiese-Rabsch; Claudia Drucker; Sven Malchow; Jürgen Scheller; Stefan Rose-John (1054-1061).
Interleukin-6 (IL-6) plays an important role in liver regeneration and protection against liver damage. In addition to IL-6 classic signaling via membrane bound receptor (mIL-6R), IL-6 signaling can also be mediated by soluble IL-6R (sIL-6R) thereby activating cells that do not express membrane bound IL-6R. This process has been named trans-signaling. IL-6 trans-signaling has been demonstrated to operate during liver regeneration. We have developed methods to specifically block or mimic IL-6 trans-signaling. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling whereas an IL-6/sIL-6R fusion protein (Hyper-IL-6) mimics IL-6 trans-signaling. Using these tools we investigate the role of IL-6 trans-signaling in CCl4 induced liver damage. Blockade of IL-6 trans-signaling during CCl4 induced liver damage led to higher liver damage, although induction of Cyp4502E1 and thus bioactivation of CCl4 was unchanged. Depletion of neutrophils resulted in reduced liver transaminase levels irrespective of IL-6 trans-signaling blockade. Furthermore, IL-6 trans-signaling was important for refilling of hepatocyte glycogen stores, which were depleted 24 h after CCl4 treatment. We conclude that IL-6 trans-signaling via the soluble IL-6R is important for the physiologic response of the liver to CCl4 induced chemical damage.►Interleukin-6 (IL-6) plays an important role in liver regeneration and protection against liver damage. In addition to IL-6 classic signaling via membrane bound receptor (mIL-6R), IL-6 signaling can also be mediated by soluble IL-6R (sIL-6R) thereby activating cells that do not express membrane bound IL-6R. This process has been named trans-signaling. ►A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling whereas an IL-6/sIL-6R fusion protein (Hyper-IL-6) mimics IL-6 trans-signaling. ►Using these tools we investigate the role of IL-6 trans-signaling in liver damage. Blockade of IL-6 trans-signaling during liver damage led to higher liver damage. ►Depletion of neutrophils resulted in reduced liver damage. ►IL-6 trans-signaling was important for refilling of hepatocyte glycogen stores, which were depleted 24 h after CCl4 treatment. ►We conclude that IL-6 trans-signaling via the soluble IL-6R is important for the physiologic response of the liver to CCl4 induced chemical damage.
Keywords: Interleukin-6; Carbon tetrachloride; Liver damage; Regeneration; IL-6 trans-signaling;
Functional analysis of F508del CFTR in native human colon by Andrea van Barneveld; Frauke Stanke; Stephanie Tamm; Benny Siebert; Gudrun Brandes; Nico Derichs; Manfred Ballmann; Sibylle Junge; Burkhard Tümmler (1062-1069).
The major cystic fibrosis mutation F508del has been classified by experiments in animal and cell culture models as a temperature-sensitive mutant defective in protein folding, processing and trafficking, but literature data on F508del CFTR maturation and function in human tissue are inconsistent. In the present study the molecular pathology of F508del CFTR was characterized in freshly excised rectal mucosa by bioelectric measurement of the basic defect and CFTR protein analysis by metabolic labelling or immunoblot. The majority of investigated F508del homozygous subjects expressed low amounts of complex-glycosylated mature F508del CFTR and low residual F508del CFTR-mediated chloride secretory activity in the rectal mucosa. The finding that some F508del CFTR escapes the ER quality control in vivo substantiates the hope that the defective processing and trafficking of F508del CFTR can be corrected by pharmacological agents.► Freshly excised CF rectal biopsies showed mature F508del CFTR. ► High variability of F508del CFTR processing in CF patients´ intestine. ► No correlation between amounts of mature F508del CFTR and chloride secretion. ► Biosynthesis of band C F508del CFTR in wild-type amounts in CF tissues.
Keywords: Cystic fibrosis; F508del CFTR protein analysis; Intestinal current measurement; Rectal biopsy;
Mutational hotspots in electron transfer flavoprotein underlie defective folding and function in multiple acyl-CoA dehydrogenase deficiency by Bárbara J. Henriques; Peter Bross; Cláudio M. Gomes (1070-1077).
We have carried out an extensive in silico analysis on 18 disease associated missense mutations found in electron transfer flavoprotein (ETF), and found that mutations fall essentially in two groups, one in which mutations affect protein folding and assembly, and another one in which mutations impair catalytic activity and disrupt interactions with partner dehydrogenases. We have further experimentally analyzed three of these mutations, ETFβ-p.Cys42Arg, ETFβ-p.Asp128Asn and ETFβ–p.Arg191Cys, which have been found in homozygous form in patients and which typify different scenarios in respect to the clinical phenotypes. The ETFβ-p.Cys42Arg mutation, related to a severe form of multiple acyl-CoA dehydrogenase deficiency (MADD), affects directly the AMP binding site and intersubunit contacts and impairs correct protein folding. The two other variations, ETFβ-p.Asp128Asn and ETFβ–p.Arg191Cys, are both associated with mild MADD, but these mutations have a different impact on ETF. Although none affects the overall α/β fold topology as shown by far-UV CD, analysis of the purified proteins shows that both have substantially decreased enzymatic activity and conformational stability. Altogether, this study combines in silico analysis of mutations with experimental data and has allowed establishing structural hotspots within the ETF fold that are useful to provide a rationale for the prediction of effects of mutations in ETF.►ETF mutations cause multiple acyl-CoA dehydrogenase deficiency. ►Mutations affect either protein folding and assembly, or catalytic activity. ►Structural hotspots within the ETF fold provide a rationale for prediction of effects of mutations.
Keywords: ETF; MADD; Molecular chaperones; Spectroscopy; Protein; Metabolic disorder;
Expression and regulation of a novel identified TNFAIP8 family is associated with diabetic nephropathy by Shuya Zhang; Yan Zhang; Xinbing Wei; Junhui Zhen; Ziying Wang; Minyong Li; Wei Miao; Hua Ding; Pengchao Du; Wenchao Zhang; Min He; Fan Yi (1078-1086).
Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) family are very recently identified proteins which share considerable sequence homology to regulate cellular and immune homeostasis. However, it is unknown whether TNFAIP8 family is expressed in the kidney and contributes to the regulation of renal functions. Therefore, the present study was designed to characterize the members of TNFAIP8 family in the kidney and to explore their possible roles in the development and progression of diabetic nephropathy. By RT-PCR and Western blot analyses, we found that all members of TNFAIP8 family were detected in the kidney. TNFAIP8 and TIPE2 expression was significantly increased in glomeruli from streptozotocin (STZ)-induced diabetic rats, and this upregulation was further confirmed in renal biopsies of diabetic patients. In in vitro study, TNFAIP8 was upregulated in response to high glucose in mesangial cells rather than podocytes. Moreover, a direct correlation was observed between expression of TNFAIP8 and mesangial cell proliferation and this regulation was associated with NADPH oxidase-mediated signaling pathway. However, we failed to observe the upregulation of TIPE2 in both mesangial cells and podocytes in response to high glucose. In conclusion, the present study addressed the role of TNFAIP8 family in diabetic nephropathy. These findings for the first time demonstrate that TNFAIP8 is one of critical components of a signal transduction pathway that links mesangial cell proliferation to diabetic renal injury.►Four members of TNFAIP8 family are expressed in the kidney. ►TNFAIP8 and TIPE2 expression is upregulated in diabetic nephropathy. ►TNFAIP8 plays a role in high glucose-induced mesangial cell proliferation. ►Regulation of TNFAIP8 expression is associated with NADPH oxidase activity.
Keywords: TNFAIP8 family; NADPH oxidase; Cell proliferation; Immune homeostasis; Diabetic nephropathy;
FTIR spectral signature of the effect of cardiotonic steroids with antitumoral properties on a prostate cancer cell line by Régis Gasper; Tatjana Mijatovic; Audrey Bénard; Allison Derenne; Robert Kiss; Erik Goormaghtigh (1087-1094).
We show in the present work that the infrared (IR) spectrum of human PC-3 prostate cancer cells exposed to anticancer drugs could offer a unique opportunity to get a fingerprint of all the major biochemical components (DNA, RNA, proteins, lipids, etc.) present in the cells and to identify with high sensitivity the signature of the metabolic changes induced by anticancer drugs.We investigated here the FTIR-related signatures of the effect of 4 structurally-related cardiotonic steroids (CS), i.e. ouabain, 19-hydroxy-2″-oxovoruscharin, hellebrin and 19-hydroxy-hellebrin on PC-3 cancer cells incubated between 0 and 36 h in the absence (control) or the presence of the CS. For each molecule a single spectral signature described the largest part of the time dependent modifications with a possible very minor second component. The spectral signatures characterizing the effects of each of the four CS were unique but very similar when compared to the signature of the effect of an intercalating anticancer drug, i.e. doxorubicin, selected as a positive reference compound in our study, suggesting a fully distinct set of cellular perturbations. The current study thus illustrates that Fourier Transform Infrared (FTIR) analyses can be used to identify, among the perturbations induced on a given cancer cell line, the features common to a group of anticancer compounds as well as features specific to every single drug.
Keywords: IR spectroscopy; Cancer; Drug; PC-3; FTIR; Cardiotonic steroid;
Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation by Stephanie L.H. Zeiger; Jennifer R. McKenzie; Jeannette N. Stankowski; Jacob A. Martin; David E. Cliffel; BethAnn McLaughlin (1095-1104).
Prior exposure to sub toxic insults can induce a powerful endogenous neuroprotective program known as ischemic preconditioning. Current models typically rely on a single stress episode to induce neuroprotection whereas the clinical reality is that patients may experience multiple transient ischemic attacks (TIAs) prior to suffering a stroke. We sought to develop a neuron-enriched preconditioning model using multiple oxygen glucose deprivation (OGD) episodes to assess the endogenous protective mechanisms neurons implement at the metabolic and cellular level. We found that neurons exposed to a five minute period of glucose deprivation recovered oxygen utilization and lactate production using novel microphysiometry techniques. Using the non-toxic and energetically favorable five minute exposure, we developed a preconditioning paradigm where neurons are exposed to this brief OGD for three consecutive days. These cells experienced a 45% greater survival following an otherwise lethal event and exhibited a longer lasting window of protection in comparison to our previous in vitro preconditioning model using a single stress. As in other models, preconditioned cells exhibited mild caspase activation, an increase in oxidized proteins and a requirement for reactive oxygen species for neuroprotection. Heat shock protein 70 was upregulated during preconditioning, yet the majority of this protein was released extracellularly. We believe coupling this neuron-enriched multi-day model with microphysiometry will allow us to assess neuronal specific real-time metabolic adaptations necessary for preconditioning.►We present a new model of preconditioning to capture the clinical reality of patients suffering multiple transient ischemic events prior to stroke. ►This series of multi-day sublethal stresses provides neuroprotection for up to 3 days. ►Real-time microphysiometry revealed rapid increases in aerobic respiratory activity in response to mild challenge. ►Neurons exposed to mild injury release large quantities of the neuroprotective chaperone HSP70. ►The metabolic and biochemical features of this model may allow us to determine real-time adaptation to ischemic stress.
Keywords: Preconditioning; Oxygen glucose deprivation; Microphysiometry; Reactive oxygen species; Caspase activation; Protein carbonyl formation; Heat shock protein 70; ATP;
The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-α production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice by Takehiko Shibata; Kisaburo Nagata; Yoshiro Kobayashi (1105-1111).
During inflammation, neutrophils infiltrate into the involved site and undergo apoptosis. Early apoptotic neutrophils are then cleared by phagocytes, leading to resolution of the inflammation, whereas if late apoptotic neutrophils are accumulated for some reason, they provoke proinflammatory responses such as TNF-α production. To determine how endogenously produced nitric oxide (NO) regulates neutrophil apoptosis and the resolution of inflammation, we compared peritoneal inflammation induced by Staphylococcus aureus bioparticles in wild type mice with that in inducible NO synthase (iNOS)-deficient ones. In this model, NO production was largely dependent on iNOS, the NO level peaking at 24 h. There were increases in the numbers of neutrophils and late apoptotic ones at 24 h in iNOS-deficient mice as compared with in wild type ones, and consequently TNF-α production at 36 h in iNOS-deficient mice. On the other hand, the administration of a NO donor to iNOS-deficient mice at 12 h decreased the numbers of neutrophils and late apoptotic ones at 24 h, and thereafter TNF-α production at 36 h. In addition, coculturing of macrophages with late apoptotic neutrophils caused TNF-α production and a NO donor inhibited the transmigration of neutrophils in a dose-dependent manner. Collectively, these results suggest a novel mechanism that endogenously produced NO suppresses neutrophil accumulation at a late stage of inflammation, thereby preventing the appearance of late apoptotic neutrophils and subsequent proinflammatory responses. ►S. aureus bioparticle induced peritoneal inflammation in WT and iNOS KO mice. ►Late apoptotic neutrophils and TNF-α production were found at a late stage in KO mice. ►A NO donor decreased the number of late apoptotic neutrophils and TNF-α production. ►TNF-α was produced upon coculturing macrophages with late apoptotic neutrophils. ►NO suppressed neutrophil migration in vitro.
Keywords: Nitric oxide; Apoptosis; Neutrophils; TNF-α; iNOS; Staphylococcus aureus bioparticles;
Elevated CSF N-acetylaspartylglutamate suggests specific molecular diagnostic abnormalities in patients with white matter diseases by Fanny Mochel; Nadège Boildieu; Julie Barritault; Catherine Sarret; Eleonore Eymard-Pierre; François Seguin; Raphael Schiffmann; Odile Boespflug-Tanguy (1112-1117).
Background: In order to identify biomarkers useful for the diagnosis of genetic white matter disorders we compared the metabolic profile of patients with leukodystrophies with a hypomyelinating or a non-hypomyelinating MRI pattern. Methods: We used a non-a priori method of in vitro 1H-NMR spectroscopy on CSF samples of 74 patients with leukodystrophies. Results: We found an elevation of CSF N-acetylaspartylglutamate (NAAG) in patients with Pelizaeus–Merzbacher disease (PMD)—PLP1 gene, Pelizaeus–Merzbacher-like disease—GJC2 gene and Canavan disease—ASPA gene. In the PMD group, NAAG was significantly elevated in the CSF of all patients with PLP1 duplication (19/19) but was strictly normal in 6 out of 7 patients with PLP1 point mutations. Additionally, we previously reported increased CSF NAAG in patients with SLC17A5 mutations. Conclusions: Elevated CSF NAAG is a biomarker that suggests specific molecular diagnostic abnormalities in patients with white matter diseases. Our findings also point to unique pathological functions of the overexpressed PLP in PMD patients with duplication of this gene.► CSF NAAG is elevated in leukodystrophies with specific molecular defects. ► NAAG is elevated in patients with PLP1 duplications but not point mutations. ► CSF NAAG can help with the molecular diagnosis of white matter disorders.
Keywords: NMR spectroscopy; N-acetylaspartylglutamate; Biomarker; Leukodystrophy; Pelizaeus–Merzbacher disease;
High-molecular weight hyaluronan reduced renal PKC activation in genetically diabetic mice by Giuseppe M. Campo; Angela Avenoso; Antonio Micali; Giancarlo Nastasi; Francesco Squadrito; Domenica Altavilla; Alessandra Bitto; Francesca Polito; Maria Grazia Rinaldi; Alberto Calatroni; Angela D'Ascola; Salvatore Campo (1118-1130).
The cluster determinant (CD44) seems to play a key role in tissues injured by diabetes type 2. CD44 stimulation activates the protein kinase C (PKC) family which in turn activates the transcriptional nuclear factor kappa B (NF-κB) responsible for the expression of the inflammation mediators such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-18 (IL-18), inducible nitric oxide synthase (iNOS), and matrix metalloproteinases (MMPs). Regulation of CD44 interaction with its ligands depends greatly upon PKC. We investigated the effect of the treatment with high-molecular weight hyaluronan (HA) on diabetic nephropathy in genetically diabetic mice.BKS.Cg-m+/+Leprdb mice had elevated plasma insulin from 15 days of age and high blood sugar levels at 4 weeks. The severe nephropathy that developed was characterized by a marked increased in CD44 receptors, protein kinase C betaI, betaII, and epsilon (PKCβI, PKCβII, and PKCε) mRNA expression and the related protein products in kidney tissue. High levels of mRNA and related protein levels were also detected in the damaged kidney for NF-κB, TNF-α, IL-6, IL-18, MMP-7, and iNOS.Chronic daily administration of high-molecular mass HA for 2 weeks significantly reduced CD44, PKCβI, PKCβII, and PKCα gene expression and the related protein production in kidney tissue and TNF-α, IL-6, IL-18, MMP-7, and iNOS expression and levels also decreased. Histological analysis confirmed the biochemical data. However, blood parameters of diabetes were unchanged.These results suggest that the CD44 and PKC play an important role in diabetes and interaction of high-molecular weight HA with these proteins may reduce inflammation and secondary pathologies due to this disease.►This study could be an additional step for a better understanding of the complex mechanism that underlies diabetes. Of particular interest is the possibility to obtain useful data for the future development of new molecules that are active in the prevention and the treatment of diabetes. ►The research in this field has made significant progress in the last years. Many studies have redefined the molecular basis of diabetes. The conventional view, although fundamental, has been supplanted by a new vision, one clearly supported by experimental and clinical data, of the diabetes disease, especially for type 2 diabetes which is age-related. In fact, it is now clear that inflammation is a key process in the onset of diabetes and that the inflammatory milieu in this detrimental disease significantly contributes to the development of many of the complications of diabetes, including diabetic nephropathy. The links between this inflammatory state and the development and progression of diabetic nephropathy involve a highly complex network of processes. Diverse inflammatory molecules play significant roles in this scenario, including CD44, PKC isoforms, chemoattractant cytokines, proinflammatory cytokines, ROS and MMPs. Consequently, on the basis of this new knowledge, a totally new approach can be planned to modulate the inflammatory mechanism that seems to underlie diabetic disease. It is widely recognised that, diabetic nephropathy is one of the worst consequences of this disease. At present, the current treatments available for diabetes complications are still suboptimal, both in terms of preventing their appearance and in halting their progression. A better understanding of the role of inflammatory molecules and processes in the context of diabetes complications will facilitate the development of new and improved therapeutic targets and strategies that can be translated successfully into clinical applications. ►It is under this new vision of the molecular basis of diabetic complications that we have performed our research study, aiming to clarify some important aspects of this disease and its damaging consequences that are currently not investigated or not well clarified. The obtained results confirmed the central role played by the CD44 receptor and the activation of PKC isoforms as well as the damage exerted by proinflammatory cytokines and the other inflammation mediators. The protective role exerted by high-molecular weight hyaluronan could be useful for future pharmacological strategies development in the treatment of diabetic complication.
Keywords: Hyaluronan; NF-κB; PKC; Cytokines; Diabetes; CD44;