BBA - Molecular Basis of Disease (v.1822, #2)

Huntington's disease (HD) is a progressive, fatal neurodegenerative disease caused by expanded polyglutamine repeats in the HD gene. HD is characterized by chorea, seizures, involuntary movements, dystonia, cognitive decline, intellectual impairment and emotional disturbances. Research into mutant huntingtin (Htt) and mitochondria has found that mutant Htt interacts with the mitochondrial protein dynamin-related protein 1 (Drp1), enhances GTPase Drp1 enzymatic activity, and causes excessive mitochondrial fragmentation and abnormal distribution, leading to defective axonal transport of mitochondria and selective synaptic degeneration. This article summarizes latest developments in HD research and focuses on the role of abnormal mitochondrial dynamics and defective axonal transport in HD neurons. This article also discusses the therapeutic strategies that decrease mitochondrial fragmentation and neuronal damage in HD.► Summarized the link between mutant Htt and mitochondrial dynamics in Huntington's disease. ► Discussed mutant Htt interaction with Drp1 in Huntington's disease neurons. ► Highlighted possible factors of defective axonal transport in Huntington's disease neurons. ► Discussed the therapeutic approaches for Huntington's disease.
Keywords: Mutant huntingtin; Abnormal mitochondrial dynamics; Defective axonal transport; RNA silencing; BACHD mouse; Mitochondrial trafficking;

Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: Implications for aging by Peizhong Mao; Patience Gallagher; Samira Nedungadi; Maria Manczak; Ulziibat P. shirendeb; Steven G. Kohama; Betsy Ferguson; Byung S. Park; P. Hemachandra Reddy (111-119).
The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.► Investigated the mtDNA deletions and mRNA expression in young, aged, male and female rhesus monkeys. ► Found increased mtDNA deletions in aged monkeys relative to young monkeys. ► mtDNA content is increased in the brains of aged monkeys, probably to compensate defective mitochondrial function. ► Discussed the compensatory aspects related to aged-dependent mitochondrial dysfunction.
Keywords: Rhesus monkeys; Mitochondrial DNA; Aging; Real-time RT-PCR; Mitochondrial DNA deletion; Mitochondrial-encoded DNA;

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function by Ram H. Nagaraj; Rooban B. Nahomi; Shilpa Shanthakumar; Mikhail Linetsky; Smitha Padmanabha; Nagarekha Pasupuleti; Benlian Wang; Puttur Santhoshkumar; Alok Kumar Panda; Ashis Biswas (120-129).
α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N ε -acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.► Lysine acetylation occurs in α-crystallin, a major protein of the human lens. ► The chaperone function of αA-crystallin is enhanced by lysine acetylation. ► The surface hydrophobicity αA-crystallin is increased by lysine acetylation. ► The acetylation mimic of αA-crystallin (K70Q) shows increased chaperone function. ► Acetylation could regulate the chaperone function of small heat shock proteins.
Keywords: Lysine; Acetylation; α-Crystallin; Chaperone; Human lens; Aging;

Association between frontal cortex oxidative damage and beta-amyloid as a function of age in Down syndrome by Giovanna Cenini; Amy L.S. Dowling; Tina L. Beckett; Eugenio Barone; Cesare Mancuso; Michael Paul Murphy; Harry LeVine; Ira T. Lott; Frederick A. Schmitt; D. Allan Butterfield; Elizabeth Head (130-138).
Down syndrome (DS) is the most common genetic cause of intellectual disability in children, and the number of adults with DS reaching old age is increasing. By the age of 40 years, virtually all people with DS have sufficient neuropathology for a postmortem diagnosis of Alzheimer disease (AD). Trisomy 21 in DS leads to an overexpression of many proteins, of which at least two are involved in oxidative stress and AD: superoxide dismutase 1 (SOD1) and amyloid precursor protein (APP). In this study, we tested the hypothesis that DS brains with neuropathological hallmarks of AD have more oxidative and nitrosative stress than those with DS but without significant AD pathology, as compared with similarly aged-matched non-DS controls. The frontal cortex was examined in 70 autopsy cases (n = 29 control and n = 41 DS). By ELISA, we quantified soluble and insoluble Aβ40 and Aβ42, as well as oligomers. Oxidative and nitrosative stress levels (protein carbonyls, 4-hydroxy-2-trans-nonenal (HNE)-bound proteins, and 3-nitrotyrosine) were measured by slot-blot. We found that soluble and insoluble amyloid beta peptide (Aβ) and oligomers increase as a function of age in DS frontal cortex. Of the oxidative stress markers, HNE-bound proteins were increased overall in DS. Protein carbonyls were correlated with Aβ40 levels. These results suggest that oxidative damage, but not nitrosative stress, may contribute to the onset and progression of AD pathogenesis in DS. Conceivably, treatment with antioxidants may provide a point of intervention to slow pathological alterations in DS.► Older people with Down syndrome are at high risk for developing Alzheimer disease. ► Aβ and oligomers increase with age. ► Hydroxynonenal-bound proteins are increased in Down syndrome. ► Protein carbonyls were correlated with Aβ40 levels. ► Oxidative damage may contribute to Alzheimer disease pathogenesis in Down syndrome.
Keywords: Alzheimer disease; 4-Hydroxy-2-nonenal; 3-Nitrotyrosine; Oligomer; Protein carbonyl; Trisomy 21;

Compromised mitochondrial complex II in models of Machado–Joseph disease by Mário N. Laço; Catarina R. Oliveira; Henry L. Paulson; A. Cristina Rego (139-149).
Machado–Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.► We evaluated mitochondrial dysfunction in models of Machado–Joseph disease. ► Expanded ataxin-3 accumulation occurs in the cytoplasm and nuclei of cells. ► Nuclear ataxin-3 aggregates are correlated to Cajal and PML body distribution. ► Expanded ataxin-3 induced an increased susceptibility to complex II inhibition. ► Machado–Joseph disease models show decreased mitochondrial complex II activity.
Keywords: Machado–Joseph disease; Ataxin-3; Protein aggregation; Mitochondria; Cell death; Succinate dehydrogenase;

Lysosomal vitamin E accumulation in Niemann–Pick type C disease by Luz Fernanda Yévenes; Andrés Klein; Juan Francisco Castro; Tamara Marín; Nancy Leal; Federico Leighton; Alejandra R. Alvarez; Silvana Zanlungo (150-160).
Niemann–Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.► α-TOH levels are increased in NPC neurons and cerebellum from NPC1 deficient mice. ► α-TOH accumulates mainly in the late endocytic pathway of NPC neurons. ► The mRNA levels of the specific α-TOH transporter, α-TTP, are decreased in the cerebellum of NPC mice. ► α-TOH content is increased in hepatic purified lysosomes from NPC1 deficient mice.
Keywords: Vitamin E; Niemann–Pick C; Cholesterol; Lysosomes;

Autoantibodies in primary Sjögren's syndrome patients induce internalization of muscarinic type 3 receptors by Meihong Jin; Sung-Min Hwang; Alexander J. Davies; Yonghwan Shin; Jun-Seok Bae; Jong-Ho Lee; Eun Bong Lee; Yeong Wook Song; Kyungpyo Park (161-167).
Objectives. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocyte infiltration into the salivary and lachrymal glands, leading to dry mouth and eyes. The presence of functional autoantibodies against muscarinic type 3 receptor (M3R) has been reported in pSS patients. However, the pathological role of anti-M3R autoantibodies in pSS salivary dysfunction remains controversial. Methods. Purified IgGs were obtained from normal (control) and primary SS patients' sera (pSS IgG). Internalization of M3R and clathrin was analyzed by biochemical assay and immunofluorescence confocal microscopy using human submandibular gland (hSMG) cells. Cytoplasmic free Ca2+ concentration ([Ca2+]i) was measured by microspectrofluorimetry. Results. Incubation of hSMG cells with pSS IgG (1 mg/ml) significantly decreased M3R expression levels at the membrane. Carbachol-induced [Ca2+]i transients (CICTs) in these cells were also inhibited by pSS IgG. In contrast to pSS IgG, control IgG had no effect on both the M3R expression level and CICTs. We found that binding of pSS IgG to M3R induces phosphorylation of the receptor, and that the pSS IgG-induced M3R internalization is prevented by the lysosomal inhibitor, chloroquine. In addition, pSS IgG decreased membrane clathrin expression, which was inhibited by atropine. Our immunofluorescence study further confirmed that pSS IgG induces a co-localization of M3R with clathrin and subsequent internalization of M3R. Conclusion. pSS IgG induces internalization of M3R partly through a clathrin-mediated pathway. The results suggest M3R internalization as a potential mechanism to explain the exocrinopathy seen in pSS patients.► SS autoantibodies (SS IgG) induce internalization of muscarinic type 3 receptors. ► SS IgG inhibits the carbachol-induced calcium increase in human salivary gland. ► Our findings may provide a potential mechanism involved in the exocrinopathy in SS.
Keywords: Clathrin; Internalization; Muscarinic type 3 receptor; Sjögren's syndrome;

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome by Lock Hock Ngu; Leo G. Nijtmans; Felix Distelmaier; Hanka Venselaar; Sjenet E. van Emst-de Vries; Mariël A.M. van den Brand; Berendien J.M. Stoltenborg; Liesbeth T. Wintjes; Peter H. Willems; Lambertus P. van den Heuvel; Jan A. Smeitink; Richard J.T. Rodenburg (168-175).
In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the KM of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the KM observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.► The clinical features of 5 mitochondrial complex I deficient NDUFS2 patients were compared. ► The pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene was studied in detail. ► The Asp446Asn mutation leads to a catalytic defect in complex I. ► Modeling shows that the Asp446Asn mutation resides near the CoQ binding site in complex I. ► The KM of complex I for CoQ is similar in all NDUFS2 patients and controls.
Keywords: Complex I; NDUFS2; Mitochondrion; Respiratory chain; Enzyme activity; Coenzyme Q;

Impaired hepatic function and central dopaminergic denervation in a rodent model of Parkinson's disease: A self-perpetuating crosstalk? by Mariapia Vairetti; Andrea Ferrigno; Vittoria Rizzo; Giulia Ambrosi; Alberto Bianchi; Plinio Richelmi; Fabio Blandini; Marie-Therese Armentero (176-184).
In Parkinson's disease (PD), aside from the central lesion, involvement of visceral organs has been proposed as part of the complex clinical picture of the disease. The issue is still poorly understood and relatively unexplored. In this study we used a classic rodent model of nigrostriatal degeneration, induced by the intrastriatal injection of 6-hydroxydopamine (6-OHDA), to investigate whether and how a PD-like central dopaminergic denervation may influence hepatic functions. Rats received an intrastriatal injection of 6-OHDA or saline (sham), and blood, cerebrospinal fluid, liver and brain samples were obtained for up to 8 weeks after surgery. Specimens were analyzed for changes in cytokine and thyroid hormone levels, as well as liver mitochondrial alterations. Hepatic mitochondria isolated from animals bearing extended nigrostriatal lesion displayed increased ROS production, while membrane potential (ΔΨ) and ATP production were significantly decreased. Reduced ATP production correlated with nigral neuronal loss. Thyroid hormone levels were significantly increased in serum of PD rats compared to sham animals while steady expression of selected cytokines was detected in all groups. Hepatic enzyme functions were comparable in all animals. Our study indicates for the first time that in a rodent model of PD, hepatic mitochondria dysfunctions arise as a consequence of nigrostriatal degeneration, and that thyroid hormone represents a key interface in this CNS-liver interaction. Liver plays a fundamental detoxifying function and a better understanding of PD-related hepatic mitochondrial alterations, which might further promote neurodegeneration, may represent an important step for the development of novel therapeutic strategies.► Visceral organs are involved in the complex clinical setting of Parkinson's disease. ► Parkinsonian rats showed significant functional hepatic mitochondrial alterations. ► Reduced ATP production in liver mitochondria correlated with dopaminergic cell loss. ► Increased serum thyroid hormone levels may affect hepatic mitochondrial function. ► The reciprocal brain–liver axis may sustain degenerative processes in Parkinson's disease.
Keywords: 6-OHDA; Liver; Mitochondrion; ATP; Thyroid hormone; Membrane potential;

Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis by Ana P. Gomes; Filipe V. Duarte; Patricia Nunes; Basil P. Hubbard; João S. Teodoro; Ana T. Varela; John G. Jones; David A. Sinclair; Carlos M. Palmeira; Anabela P. Rolo (185-195).
Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance.► BBR protects against HFD-induced insulin resistance. ► BBR protects mitochondrial function and biogenesis in models of insulin resistance. ► BBR effects on mitochondrial function and biogenesis are dependent on SIRT1. ► BBR activation of AMPK is mediated by SIRT1.
Keywords: Metabolic syndrome; Berberine; Mitochondria; SIRT1; AMPK; NAMPT;

Intestinal mucosal barrier is the first line of defense against bacteria and their products originating from the intestinal lumen. We have shown a role for IL-18 in impaired gut barrier function following acute alcohol (EtOH) intoxication combined with burn injury. To further delineate the mechanism, this study examined whether IL-18 alters intestine tight junction proteins or induces mucosal apoptosis under these conditions. To accomplish this, rats were gavaged with EtOH (3.2 g/kg) prior to ~ 12.5% total body surface area burn or sham injury. One day after injury, EtOH combined with burn injury resulted in a significant decrease in total occludin protein and its phosphorylation in small intestine compared to either EtOH or burn injury alone. There was no change in claudin-1 protein content but its phosphorylation on tyrosine was decreased following EtOH and burn injury. This was accompanied with an increase in mucosal apoptosis (p < 0.05). The treatment of rats with anti-IL-18 antibody at the time of burn injury prevented intestine apoptosis and normalized tight junction proteins following EtOH and burn injury. Altogether, these findings suggest that IL-18 modulates tight junction proteins and cause apoptosis leading to impaired intestinal mucosal integrity following EtOH intoxication combined with burn injury.► We found that IL-18 modulates tight junction proteins after alcohol and burn injury. ► IL-18 also causes an increase in intestinal apoptosis after alcohol and burn injury. ► IL-18 disrupts gut barrier function following alcohol and burn injury.
Keywords: Thermal injury; Inflammatory mediator; Gut barrier; Tissue damage; Ethanol; Cytokine;

Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils.► Annexins A1 and A4 were detected on late early apoptotic HeLa cells and both early and late apoptotic human neutrophils. ► Mouse macrophages produced MIP-2 in response to late apoptotic human neutrophils, but not HeLa cells. ► Annexins A1 and A4 suppress MIP-2 or IL-8 production during coculturing of mouse macrophages with late apoptotic human neutrophils.
Keywords: Annexins A1 and A4; Early and late apoptosis; HeLa cell; Human neutrophil; Macrophage; MIP-2 or IL-8;

Changes in mitochondrial glutathione levels and protein thiol oxidation in ∆yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia by A.L. Bulteau; S. Planamente; L. Jornea; A. Dur; E. Lesuisse; J.M. Camadro; F. Auchère (212-225).
Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron–sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the •SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ∆yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.► Decrease in mitochondrial GSH/GSSG ratio in Δyfh1 yeast cells and the lymphoblasts of patients with FRDA. ► Increase in glutathione–protein adducts in both yeast and human frataxin-deficient cells. ► Thiol oxidation of key mitochondrial proteins, such as KGDH and subunits of respiratory chain complexes III and IV. ► Existence of a regulatory process involving both iron and glutathione in yeast and human cells.
Keywords: Friedreich's ataxia; Glutathione; Iron; Mitochondrion; Thiol oxidation; Protein glutathionylation;

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy by Giovana B. Biancini; Camila S. Vanzin; Daiane B. Rodrigues; Marion Deon; Graziela S. Ribas; Alethéa G. Barschak; Vanusa Manfredini; Cristina B.O. Netto; Laura B. Jardim; Roberto Giugliani; Carmen R. Vargas (226-232).
Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n = 14) and healthy age-matched controls (n = 14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.► We tested urine and blood samples of treated Fabry patients and compared to controls. ► Patients presented higher levels of pro-inflammatory and pro-oxidant biomarkers. ► Oxidative stress and inflammation were directly correlated with globotriaosylceramide. ► Globotriaosylceramide accumulated seems to induce these findings in vivo.
Keywords: Fabry disease; Oxidative stress; Globotriaosylceramide; Inflammation; Antioxidant defense; Reactive species;

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H2O2. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H2O2-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H2O2 in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H2O2 was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H2O2-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H2O2-treated normal skin fibroblasts to oxidative stress.► We showed that metabolic shift from respiration to glycolysis is an adaptive response of human cells to oxidative stress. ► Oxidative stress induced AMPK activation and phosphorylation of phosphofructokinase 2 (PFK2) to activate PFK1 and glycolysis. ► AMPK-mediated increase of glycolytic flux contributed to NADPH production for cell survival under oxidative stress. ► This scenario also occurred in the skin fibroblasts of patients with mitochondrial diseases such as MERRF syndrome.
Keywords: Mitochondrial disease; MERRF syndrome; Oxidative stress; AMPK; Glycolysis; NADPH;

MicroRNA 21 (miR-21) has been implicated in various aspects of carcinogenesis. However, its function and molecular mechanism in cervical squamous carcinoma have not been studied. Using TaqMan quantitative real-time PCR and Northern blot, we confirmed that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. Remarkably, we showed that the level of miR-21 correlates with the tumor differentiation and nodal status by ISH. Furthermore, we demonstrated that miR-21 regulates proliferation, apoptosis, and migration of HPV16-positive cervical squamous cells. In order to identify candidate target genes for miR-21, we used gene expression profiling. By luciferase reporter assays, we confirmed that CCL20 is one of its target genes, which is related to the HPV16 E6 and E7 oncogenes. Our results suggest that miR-21 may be involved in cervical squamous cell tumorigenesis.► We confirmed the overexpression of miR-21 in human cervical squamous cancer and the level of miR-21 correlating with the nodal status and differentiation. ► We demonstrated that miR-21 may involve in cervical squamous cells proliferation, apoptosis, and migration with HPV16. ► By bioinformatic analysis and luciferase activity assays, we proved that CCL20 contains putative miR-21 binding sites.
Keywords: Cervical squamous carcinoma; Cervical intra-epithelial neoplasia; Human papillomavirus; MIR21; CCL20;

α-Synuclein misfolding and Parkinson's disease by Leonid Breydo; Jessica W. Wu; Vladimir N. Uversky (261-285).
Substantial evidence links α-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic Parkinson's disease (PD). α-Synuclein has been identified as the major component of Lewy bodies and Lewy neurites, the characteristic proteinaceous deposits that are the hallmarks of PD. α-Synuclein is a typical intrinsically disordered protein, but can adopt a number of different conformational states depending on conditions and cofactors. These include the helical membrane-bound form, a partially-folded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. The molecular basis of PD appears to be tightly coupled to the aggregation of α-synuclein and the factors that affect its conformation. This review examines the different aggregation states of α-synuclein, the molecular mechanism of its aggregation, and the influence of environmental and genetic factors on this process.► α-Synuclein misfolding and aggregation are linked to the Parkinson's disease pathology. ► In the unbound form, α-synuclein is a typical intrinsically disordered protein. ► It can adopt different conformations depending on the environmental modulators. ► Many environmental factors promote α-synuclein misfolding and aggregation. ► Structural variability of aggregated forms correlates with their effects in vivo.
Keywords: α-Synuclein; Parkinson's disease; Neurodegeneration; Aggregation; Intrinsically disordered protein; Metal-exposure;

Multi-faced neuroprotective effects of Ginsenoside Rg1 in an Alzheimer mouse model by Fang Fang; Xiaochun Chen; Tianwen Huang; Lih-Fen Lue; John S. Luddy; Shirley ShiDu Yan (286-292).
There has been no extensive characterization of the effects of Ginsenoside Rg1, a pharmacological active component purified from the nature product ginseng, in an Alzheimer's disease mouse model. The well-characterized transgenic Alzheimer disease (AD) mice over expressing amyloid precursor protein (APP)/Aβ (Tg mAPP) and nontransgenic (nonTg) littermates at age of 6 and 9 months were treated with Rg 1 for three months via intraperitoneal injection. Mice were then evaluated for changes in amyloid pathology, neuropathology and behavior. Tg mAPP treated with Rg1 showed a significant reduction of cerebral Aβ levels, reversal of certain neuropathological changes, and preservation of spatial learning and memory, as compared to vehicle-treated mice. Rg1 treatment inhibited activity of γ-secretase in both Tg mAPP mice and B103-APP cells, indicating the involvement of Rg1 in APP regulation pathway. Furthermore, administration of Rg1 enhanced PKA/CREB pathway activation in mAPP mice and in cultured cortical neurons exposed to Aβ or glutamate-mediated synaptic stress. Most importantly, the beneficial effects on attenuation of cerebral Aβ accumulation, improvement in neuropathological and behavioral changes can be extended to the aged mAPP mice, even to 12–13 months old mice that had extensive amyloid pathology and severe neuropathological and cognitive malfunction. These studies indicate that Rg1 has profound multi-faced and neuroprotective effects in an AD mouse model. Rg1 induces neuroprotection through ameliorating amyloid pathology, modulating APP process, improving cognition, and activating PKA/CREB signaling. These findings provide a new perspective for the treatment of AD and demonstrate potential for a new class of drugs for AD treatment.► Rg1 treatment inhibits activity of γ-secretase in transgenic mAPP mice. ► Rg1 significantly reduced cerebral Aβ accumulation. ► Administration of Rg1 improves learning/memory and reverses neuropathological changes.
Keywords: Rg1; AD mouse model; Neuroprotection; Gamma secretase;

Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype by Jana Jandova; Mingjian Shi; Kimberly G. Norman; George P. Stricklin; James E. Sligh (293-300).
There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse ρ0 cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.► Cybrids are useful models to study the role of mtDNA changes in cancer development. ► Somatic mutations in mtDNA produce changes in cellular growth and motility. ► There are mtDNA-driven differences in production of intracellular ROS. ► Antioxidants were able to diminish the tumorigenic phenotypes of mutant cybrids. ► Mutations in mtDNA play important roles in the development of neoplasia.
Keywords: mtDNA mutation; Proliferation; Reactive oxygen species; Antioxidants; UV-induced apoptosis; Migration and invasion;

Up-regulation of NDRG2 through nuclear factor-kappa B is required for Leydig cell apoptosis in both human and murine infertile testes by Teng Li; Jing Hu; Gong-Hao He; Yun Li; Chu-Chao Zhu; Wu-Gang Hou; Shun Zhang; Wei Li; Jin-Shan Zhang; Zhe Wang; Xin-Ping Liu; Li-Bo Yao; Yuan-Qiang Zhang (301-313).
Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-κB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague–Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-κB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility.►NDRG2 and NF-κB expression was characterized in apoptotic cells of human and murine infertile testes. ►NF-κB was activated and binds to ndrg2 gene promoter, up-regulate NDRG2 in apoptotic Leydig cells. ►Inhibition of NF-κB through PDTC blocked EDS induced Leydig cell apoptosis in vivo and in vitro. ►Knock-down of NDRG2 by siRNA attenuates EDS induced Leydig cell apoptosis in vitro. ►Over-expression of NDRG2 by plasmid transfection triggers EDS induced Leydig cell apoptosis in vitro.
Keywords: Nuclear factor-kappa B; N-myc downstream-regulated gene 2; Infertile disease; Leydig cell; Apoptosis; Pyrrolidine dithiocarbamate;