Irène Ceballos-Picot

Glutathione antioxidant system as a marker of oxidative stress in chronic renal failure

A profound imbalance between oxidants and antioxidants has been suggested in uremic patients on maintenance hemodialysis. However, the respective influence of uremia and dialysis procedure has not been evaluated. Circulating levels of copper-zinc superoxide dismutase (CuZn SOD), glutathione peroxidase (GSH-Px), and reductase (GSSG-Rd), total GSH and GSSG were determined in a large cohort of 233 uremic patients including 185 undialyzed patients with mild to severe chronic renal failure, and 48 patients treated by peritoneal dialysis or hemodialysis. Compared to controls, erythrocyte GSH-Px and GSSG-Rd activities were significantly increased at the mild stage of chronic uremia (p < .001), whereas erythrocyte CuZn SOD activity was unchanged, total level of GSH and plasma GSH-Px activity were significantly decreased, and GSSG level and GSSG-Rd activity were unchanged. Positive Spearman rank correlations were observed between creatinine clearance and plasma levels of GSH-Px (r = .65, p < .001), selenium (r = .47, p < .001), and GSH (r = .41, p < .001). Alterations in antioxidant systems gradually increased with the degree of renal failure, further rose in patients on peritoneal dialysis and culminated in hemodialysis patients in whom an almost complete abolishment of GSH-Px activity was observed. In conclusion, such disturbances in antioxidant systems that occur from the early stage of chronic uremia and are exacerbated by dialysis provide additional evidence for a resulting oxidative stress that could contribute to the development of accelerated atherosclerosis and other long-term complications in uremic patients.

Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid peroxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47; P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 +/- 0.71 mumole MDA/g tissue) than at 2 months (5.69 +/- 0.90) and significantly lower at 28 months (5.13 +/- 0.33) than at 12 months. Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.

Attenuated variants of Lesch-Nyhan disease

Lesch–Nyhan disease is a neurogenetic disorder caused by deficiency of the enzyme hypoxanthine–guanine phosphoribosyltransferase. The classic form of the disease is described by a characteristic syndrome that includes overproduction of uric acid, severe generalized dystonia, cognitive disability and self-injurious behaviour. In addition to the classic disease, variant forms of the disease occur wherein some clinical features are absent or unusually mild. The current studies provide the results of a prospective and multi-centre international study focusing on neurological manifestations of the largest cohort of Lesch–Nyhan disease variants evaluated to date, with 46 patients from 3 to 65 years of age coming from 34 families. All had evidence for overproduction of uric acid. Motor abnormalities were evident in 42 (91%), ranging from subtle clumsiness to severely disabling generalized dystonia. Cognitive function was affected in 31 (67%) but it was never severe. Though none exhibited self-injurious behaviours, many exhibited behaviours that were maladaptive. Only three patients had no evidence of neurological dysfunction. Our results were compared with a comprehensive review of 78 prior reports describing a total of 127 Lesch–Nyhan disease variants. Together these results define the spectrum of clinical features associated with hypoxanthine–guanine phosphoribosyltransferase deficiency. At one end of the spectrum are patients with classic Lesch–Nyhan disease and the full clinical phenotype. At the other end of the spectrum are patients with overproduction of uric acid but no apparent neurological or behavioural deficits. Inbetween are patients with varying degrees of motor, cognitive, or behavioural abnormalities. Recognition of this spectrum is valuable for understanding the pathogenesis and diagnosis of all forms of hypoxanthine–guanine phosphoribosyltransferase deficiency.

Peripheral antioxidant enzyme activities and selenium in elderly subjects and in dementia of Alzheimer's type—Place of the extracellular glutathione peroxidase

Defenses against free radical damage were determined in red blood cells and plasma from 40 patients with dementia of the Alzheimer-type (DAT) and 34 aged control subjects with normal cognitive function. No crude significant difference in erythrocyte copper-zinc superoxide dismutase (E-CuZnSOD), seleno-dependent glutathione peroxidase (E-GSH-Px), glutathione reductase (E-GSSG-RD) activities, and selenium (Se) concentration was found between DAT cases and control subjects. The peroxidation products evaluated in plasma by the thiobarbituric-reactive material (TBARS) were at the same level in the DAT group as compared to controls. In the DAT group, plasma GSH-Px (P-GSH-Px) activity and plasma Se (P-Se) were negatively correlated with age (r = -0.58; p < 0.001 and r = -0.63; p < 0.001 respectively). Moreover, erythrocyte GSH-Px activity and Se were also negatively correlated with age (r = -0.40; p < 0.01 and r = -0.46; p < 0.01, respectively). No significant correlation with age was observed in the controls. When controlling for age, a significant increase for P-GSH-Px activity and P-Se was observed in DAT patients as compared to controls. These significant differences mostly appeared in DAT subjects under 80 years. Some correlations were only observed in the DAT group such as P-GSH-Px and E-GSH-Px (r = +0.68; p < 0.001); P-GSH-Px and E-Se (r = +0.79; p < 0.001). Correlations between P-GSH-Px and P-Se, E-GSH-Px and P-Se, and P-Se with E-Se are greater in the DAT group (r = +0.84; p < 0.001; r = +0.76; p < 0.001 and r = 0.75; p < 0.001) than in the control group (r = 0.54, pI < 0.01; r = 0.43, p < 0.01 and r = +0.34, p < 0.05 respectively). The fact that first -- a significant increase in P-GSH-Px and P-Se, second -- some modifications in the relationships between antioxidant parameters, and third -- age-dependent decreases of glutathione-peroxidase activities and their cofactor, appeared only in the DAT group suggest that DAT is associated with an oxidative stress due to an imbalance between reactive oxygen species and the peripheral antioxidant opposing forces.

Overproduction of Cu/Zn-superoxide dismutase or Bcl-2 prevents the brain mitochondrial respiratory dysfunction induced by glutathione depletion

Recent work has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Low concentrations of the powerful antioxidant glutathione (GSH) and impaired brain energy metabolism, particularly in the substantia nigra, are key features of Parkinsons disease (PD). The main goal of this study was to better characterize the deleterious effects of brain GSH depletion on mitochondrial function. We depleted GSH in the brains of newborn wild-type (WT) and transgenic (Tg) mice overproducing either human Cu/Zn-superoxide dismutase (h-CuZnSOD) or human Bcl2 (h-Bcl-2), by subcutaneous injection of l-buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. GSH was 97% depleted in brain homogenates and 90% depleted in brain mitochondria for both WT and Tg mice. This depletion of brain GSH led to a decrease in the activity of the GSH-dependent antioxidant enzyme glutathione peroxidase, both in WT and in Tg animals. BSO treatment decreased the activities of respiratory complexes I, II, and IV in the brain homogenates of WT mice. BSO-treated h-CuZnSOD or h-Bcl-2 Tg mice had no respiratory chain deficiencies. Thus, brain GSH depletion leads to the impairment of mitochondrial respiratory chain activity. The protection of mitochondrial respiratory function by overproduction of Bcl-2 may result from a decrease in the generation of reactive oxygen species (ROS) or lipid peroxidation. The protection of mitochondria by overproduction of CuZnSOD is consistent with the involvement of superoxide or superoxide-derived ROS in the mitochondrial dysfunction caused by brain GSH depletion. This study demonstrates that the antioxidant balance is critical for maintenance of brain mitochondrial function, and its disruption may contribute to the pathogenesis of PD.

Genotype-phenotype correlations in neurogenetics: Lesch-Nyhan disease as a model disorder

Establishing meaningful relationships between genetic variations and clinical disease is a fundamental goal for all human genetic disorders. However, these genotype-phenotype correlations remain incompletely characterized and sometimes conflicting for many diseases. Lesch-Nyhan disease is an X-linked recessive disorder that is caused by a wide variety of mutations in the HPRT1 gene. The gene encodes hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in purine metabolism. The fine structure of enzyme has been established by crystallography studies, and its function can be measured with very precise biochemical assays. This rich knowledge of genetic alterations in the gene and their functional effect on its protein product provides a powerful model for exploring factors that influence genotype-phenotype correlations. The present study summarizes 615 known genetic mutations, their influence on the gene product, and their relationship to the clinical phenotype. In general, the results are compatible with the concept that the overall severity of the disease depends on how mutations ultimately influence enzyme activity. However, careful evaluation of exceptions to this concept point to several additional genetic and non-genetic factors that influence genotype-phenotype correlations. These factors are not unique to Lesch-Nyhan disease, and are relevant to most other genetic diseases. The disease therefore serves as a valuable model for understanding the challenges associated with establishing genotype-phenotype correlations for other disorders.

MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy

MEDNIK syndrome-acronym for mental retardation, enteropathy, deafness, neuropathy, ichthyosis, keratodermia-is caused by AP1S1 gene mutations, encoding σ1A, the small subunit of the adaptor protein 1 complex, which plays a crucial role in clathrin coat assembly and mediates trafficking between trans-Golgi network, endosomes and the plasma membrane. MEDNIK syndrome was first reported in a few French-Canadian families sharing common ancestors, presenting a complex neurocutaneous phenotype, but its pathogenesis is not completely understood. A Sephardic-Jewish patient, carrying a new AP1S1 homozygous mutation, showed severe perturbations of copper metabolism with hypocupremia, hypoceruloplasminemia and liver copper accumulation, along with intrahepatic cholestasis. Zinc acetate treatment strikingly improved clinical conditions, as well as liver copper and bile-acid overload. We evaluated copper-related metabolites and liver function retrospectively in the original French-Canadian patient series. Intracellular copper metabolism and subcellular localization and function of copper pump ATP7A were investigated in patient fibroblasts. Copper metabolism perturbation and hepatopathy were confirmed in all patients. Studies in mutant fibroblasts showed abnormal copper incorporation and retention, reduced expression of copper-dependent enzymes cytochrome-c-oxidase and Cu/Zn superoxide dismutase, and aberrant intracellular trafficking of Menkes protein ATP7A, which normalized after rescue experiments expressing wild-type AP1S1 gene. We solved the pathogenetic mechanism of MEDNIK syndrome, demonstrating that AP1S1 regulates intracellular copper machinery mediated by copper-pump proteins. This multisystem disease is characterized by a unique picture, combining clinical and biochemical signs of both Menkes and Wilsons diseases, in which liver copper overload is treatable by zinc acetate therapy, and can now be listed as a copper metabolism defect in humans. Our results may also contribute to understand the mechanism(s) of intracellular trafficking of copper pumps.

Hypoxanthine-guanine phosphoribosyl transferase regulates early developmental programming of dopamine neurons: implications for Lesch-Nyhan disease pathogenesis

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in Lesch-Nyhan disease (LND), where affected individuals exhibit a characteristic neurobehavioral disorder that has been linked with dysfunction of dopaminergic pathways of the basal ganglia. Since the functions of HPRT, a housekeeping enzyme responsible for recycling purines, have no direct relationships with the dopaminergic pathways, the mechanisms whereby HPRT deficiency affect them remain unknown. The current studies demonstrate that HPRT deficiency influences early developmental processes controlling the dopaminergic phenotype, using several different cell models for HPRT deficiency. Microarray methods and quantitative PCR were applied to 10 different HPRT-deficient (HPRT(-)) sublines derived from the MN9D cell line. Despite the variation inherent in such mutant sublines, several consistent abnormalities were evident. Most notable were increases in the mRNAs for engrailed 1 and 2, transcription factors known to play a key role in the specification and survival of dopamine neurons. The increases in mRNAs were accompanied by increases in engrailed proteins, and restoration of HPRT reverted engrailed expression towards normal levels, demonstrating a functional relationship between HPRT and engrailed. The functional relevance of the abnormal developmental molecular signature of the HPRT(-) MN9D cells was evident in impoverished neurite outgrowth when the cells were forced to differentiate chemically. To verify that these abnormalities were not idiosyncratic to the MN9D line, HPRT(-) sublines from the SK-N-BE(2) M17 human neuroblastoma line were evaluated and an increased expression of engrailed mRNAs was also seen. Over-expression of engrailed occurred even in primary fibroblasts from patients with LND in a manner that suggested a correlation with disease severity. These results provide novel evidence that HPRT deficiency may affect dopaminergic neurons by influencing early developmental mechanisms.

Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder causing 2,8-dihydroxyadenine stones and renal failure secondary to intratubular crystalline precipitation. Little is known regarding the clinical presentation of APRT deficiency, especially in the white population. We retrospectively reviewed all 53 cases of APRT deficiency (from 43 families) identified at a single institution between 1978 and 2009. The median age at diagnosis was 36.3 years (range 0.5 to 78.0 years). In many patients, a several-year delay separated the onset of symptoms and diagnosis. Of the 40 patients from 33 families with full clinical data available, 14 (35%) had decreased renal function at diagnosis. Diagnosis occurred in six (15%) patients after reaching ESRD, with five diagnoses made at the time of disease recurrence in a renal allograft. Eight (20%) patients reached ESRD during a median follow-up of 74 months. Thirty-one families underwent APRT sequencing, which identified 54 (87%) mutant alleles on the 62 chromosomes analyzed. We identified 18 distinct mutations. A single T insertion in a splice donor site in intron 4 (IVS4 + 2insT), which produces a truncated protein, accounted for 40.3% of the mutations. We detected the IVS4 + 2insT mutation in two (0.98%) of 204 chromosomes of healthy newborns. This report, which is the largest published series of APRT deficiency to date, highlights the underdiagnosis and potential severity of this disease. Early diagnosis is crucial for initiation of effective treatment with allopurinol and for prevention of renal complications.

Selenium and Oxygen‐Metabolizing Enzymes in Elderly Community Residents: A Pilot Epidemiological Study

To investigate the relationship of selenium and oxygen‐deactivating enzymes with age in the elderly.