BBA - Molecular Basis of Disease (v.1812, #6)

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells by Martijn J. Wilmer; Leo A.J. Kluijtmans; Thea J. van der Velden; Peter H. Willems; Peter G. Scheffer; Rosalinde Masereeuw; Leo A. Monnens; Lambertus P. van den Heuvel; Elena N. Levtchenko (643-651).
Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.► Presentation of a new human cystinosis renal proximal tubular cell model. ► Altered GSSG/2GSH redox status in cystinosis cells. ► Energy generating capacity is normal in cystinosis cells, but decreased ATP levels are found. ► Treatment with cysteamine restores redox status in vitro via increased GSH levels. ► Restoration of redox status by cysteamine might be beneficial in other renal disorders.
Keywords: Cystinosis; Cysteamine; Glutathione; Oxidative stress; Redox status; ATP production;

ETS-domain transcription factor Elk-1 mediates neuronal survival: SMN as a potential target by Ozlem Demir; Nese Aysit; Zeynep Onder; Nezaket Turkel; Gurkan Ozturk; Andrew D. Sharrocks; Isil Aksan Kurnaz (652-662).
Elk-1 belongs to the ternary complex factors (TCFs) subfamily of the ETS domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with serum response elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAi-mediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-of-motor neuron-1 (SMN1) gene as a novel target of Elk-1, and show that the ets motifs in the SMN1 promoter are involved in this regulation.Elk-1 mediates survival in different neuronal model cells ► RNAi of Elk-1 results in enhanced apoptosis ► Elk-1 can regulate Mcl-1 in neuronal models ► Elk-1 can bind to and regulate SMN promoter
Keywords: Neuronal apoptosis; Neuroprotection; SMA; SMN; Survival; Transcription factor;

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease by Sirsendu Jana; Maitrayee Sinha; Dalia Chanda; Tapasi Roy; Kalpita Banerjee; Soumyabrata Munshi; Birija S. Patro; Sasanka Chakrabarti (663-673).
The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100–400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.► Dopamine inhibits mitochondrial oxidative phosphorylation. ► Mitochondrial dysfunction is mediated by dopamine derived quinones. ► In PC12 cells dopamine induces apoptosis through mitochondrial impairment. ► Quinone scavengers can have neuroprotective potential in Parkinson's disease.
Keywords: Dopamine; Parkinson's disease; Mitochondria; Quinone; Apoptosis; Oxidative stress;

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons by Hung-Li Wang; An-Hsun Chou; Ai-Shun Wu; Si-Ying Chen; Yi-Hsin Weng; Yu-Cheng Kao; Tu-Hsueh Yeh; Po-Ju Chu; Chin-Song Lu (674-684).
Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (ΔΨ m) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized ΔΨ m. In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H2O2-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized ΔΨ m and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal ΔΨ m and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.► PINK1-null SN dopaminergic neurons exhibited depolarized mitochondrial potential. ► PINK1-deficient SN dopaminergic neurons displayed mitochondrial fragmentation. ► Overexpression of wild-type PINK1 restored mitochondrial potential and morphology. ► PARK6 PINK1 mutants failed to restore mitochondrial function and morphology.
Keywords: Parkinson's disease; PTEN-induced kinase 1; Substantia nigra dopaminergic neuron; Mitochondrial membrane potential; Mitochondrial fragmentation; Reactive oxygen species;

The molecular basis of aminoacylase 1 deficiency by Anke Sommer; Ernst Christensen; Susanne Schwenger; Ralf Seul; Dorothea Haas; Heike Olbrich; Heymut Omran; Jörn Oliver Sass (685-690).
Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.► Clinical and biochemical data on aminoacylase 1 (ACY1) deficiency are expanded. ► Transient overexpression of all known mutations underlying ACY1 deficiency. ► Most mutations result in nearly complete loss of enzyme function. ► Newly discovered mutations yield considerable residual activity of the enzyme. ► Intramolecular localizations and molecular characteristics can explain residual activities.
Keywords: Amidohydrolase; Organic acid; Inborn error of metabolism; N-acetyl amino acid; N-acetyl methionine; N-acetyl glutamic acid;

Clinical and diagnostic approach in unsolved CDG patients with a type 2 transferrin pattern by M. Mohamed; M. Guillard; S.B. Wortmann; S. Cirak; E. Marklova; H. Michelakakis; E. Korsch; M. Adamowicz; B. Koletzko; F.J. van Spronsen; K.E. Niezen-Koning; G. Matthijs; T. Gardeitchik; D. Kouwenberg; B. Chan Lim; R. Zeevaert; R.A. Wevers; D.J. Lefeber; E. Morava (691-698).
Dysmorphic features, multisystem disease, and central nervous system involvement are common symptoms in congenital disorders of glycosylation, including several recently discovered Golgi-related glycosylation defects. In search for discriminative features, we assessed eleven children suspected with a Golgi-related inborn error of glycosylation. We evaluated all genetically unsolved patients, diagnosed with a type 2 transferrin isofocusing pattern in the period of 1999–2009. By combining biochemical results with characteristic clinical symptoms, we used a diagnostic flow chart to approach the underlying defect in patients with congenital disorders of glycosylation-IIx. According to specific symptoms and laboratory results, we initiated additional, targeted biochemical and genetic studies. We found a distinctive spectrum of congenital disorders of glycosylation type 2-associated anomalies including sudden hearing loss, brain malformations, wrinkled skin, and epilepsy in combination with skeletal dysplasia, dilated cardiomyopathy, sudden cardiac arrest, abnormal copper and iron metabolism, and endocrine abnormalities in our patients. One patient with severe cortical malformations and mild skin abnormalities was diagnosed with a known genetic syndrome, due to an ATP6V0A2 defect. Here, we present unique congenital disorders of glycosylation type 2-associated anomalies, including both ATPase-related and unrelated cutis laxa and sensorineural hearing loss, a recently recognized symptom of congenital disorders of glycosylation. Based on our findings, we recommend clinicians to consider congenital disorders of glycosylation in patients with cardiac rhythm disorders, spondylodysplasia and biochemical abnormalities of the copper and iron metabolism even in absence of intellectual disability.►The genetic etiology in most CDG patients with type 2 TIEF pattern remains unknown. ►We systematically evaluate 11 unsolved patients and define discriminative clinical symptoms. ►We use an extended biochemical diagnostic “tool-kit” in CDG-IIx. ►We report on unique laboratory findings in lysosomal function and copper and iron metabolism. ►Deafness and non ATPase-related cutis laxa are also observed in CDG-IIx.
Keywords: CDG type 2; CDG-IIx; Golgi-system; Hearing loss; Seizure; TIEF; Copper metabolism;

Early reduction of circulating homocysteine levels in Goto–Kakizaki rat, a spontaneous nonobese model of type 2 diabetes by Christophe Noll; Gregory Lacraz; Jan Ehses; Josiane Coulaud; Danièle Bailbe; Jean-Louis Paul; Bernard Portha; Françoise Homo-Delarche; Nathalie Janel (699-702).
Diabetes mellitus is associated with increased risk for cardiovascular disorders, which are major causes of mortality in this disease. Hyperhomocysteinemia, defined by high plasma homocysteine levels, is an independent risk factor for the development of cardiovascular diseases. Type 2 diabetic patients have higher circulating homocysteine levels than healthy subjects and these levels are even higher in plasma of obese than nonobese diabetic patients. Homocysteine metabolism that has been studied in 2 animal models of type 2 diabetes with obesity led to conflicting data. The aim of the present study was to analyze homocysteine metabolism in a spontaneous nonobese model of type 2 diabetes, the Goto–Kakizaki rats at various successive and well characterized stages of the disease: during early postnatal normoglycemia, at the onset of hyperglycemia (around weaning), and during chronic mild hyperglycemia with progressive insulin resistance. Compared to age-matched Wistar controls, Goto–Kakizaki rats showed lower plasma levels of homocysteine and a falling trend in its major byproduct antioxidant, glutathione, from the prediabetic stage onwards. Concomitantly, Goto–Kakizaki rats exhibited increased liver activity of cystathionine beta synthase, which catalyzes the condensation of homocysteine with serine in the first step of the transsulfuration pathway. These results emphasize a strong association between homocysteine metabolism and insulin via the first step of the hepatic transsulfuration pathway in Goto–Kakizaki rats.► GK rats have lower plasma homocysteine level. ► GK rats have lower plasma glutathione level. ► Cystathionine beta synthase activity is increased in liver of GK rats.
Keywords: Homocysteine; Cystathionine beta synthase; Glutathione; Type 2 diabetes; Goto–Kakizaki rat; Prediabetes;

Identification of Dermcidin as a novel binding protein of Nck1 and characterization of its role in promoting cell migration by Shun-Li Shen; Fang-Hua Qiu; Thamara K. Dayarathna; Jian Wu; Ming Kuang; Shawn S.-C. Li; Bao-Gang Peng; Jing Nie (703-710).
A distinct feature of hepatocellular carcinoma (HCC) is the tendency of tumor cells to disperse throughout the liver. Nck family adaptor proteins function to couple tyrosine phosphorylation signals to regulate actin cytoskeletal reorganization that leads to cell motility. In order to explore the role of Nck in HCC development, we performed GST pull-down assay using the SH2 domain of Nck1 as bait. The resulting precipitates were separated by 2-DE. Mass spectrometry analysis revealed a group of Nck1 SH2 domain-binding proteins that were differentially expressed in HCC. One of these proteins, dermcidin (DCD), and its interaction with Nck1, was further validated in vitro. GST pull-down assay revealed that Nck1 SH2 domain binds to the phosphotyrosine residue at position 20 (Y20) of the DCD. Pervandate treatment significantly enhanced the interaction between DCD and Nck1. Moreover, we demonstrated that forced expression of DCD could activate Rac1 and Cdc42 and promoted cell migration. Taken together, these data suggest a role of DCD in tumor metastasis.► Expression of DCD is significantly increased in HCC tissues. ► Interaction between DCD and Nck1 is tyrosine phosphorylation-dependent. ► DCD promotes migration of tumor cells via interaction with Nck1.
Keywords: Dermcidin; Nck1; SH2 domain; Cell migration; Hepatocellular carcinoma; Tyrosine phosphorylation;