Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by dual activation of the ras-phosphoinositide 3-kinase and jun n-terminal kinase pathways.

Glutaredoxin 2 (Grx2) from Escherichia coli protects cerebellar neurons from dopamine-induced apoptosis via nuclear factor kappa B (NF-kappaB) activation, which is mediated by the expression of redox factor-1 (Ref-1). An analysis of the mechanisms underlying Grx2 protective activity revealed dual activation of signal transduction pathways. Grx2 significantly activated the Ras/phosphoinositide 3-kinase/Akt/NF-kappaB cascade in parallel to the Jun N-terminal kinase (JNK)/AP1 cascade. Dopamine, in comparison, down-regulated both pathways. Treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate Akt and AP-1 but had no effect on the phosphorylation of JNK1/2, suggesting that Akt/NF-kappaB and AP-1 are regulated by Ref-1. Exposure of the neurons to JNK1/2 antisense oligonucleotide in the presence of Grx2 significantly reduced AP-1 and NF-kappaB DNA binding activities and abolished Grx2 protection. These results demonstrate that dual activation of Ras/phosphoinositide 3-kinase and AP-1 cascades, which are mediated by Ref-1, is an essential component of the Grx2 mechanism of action.

Activation of nuclear transcription factor kappa B (NF-kappaB) is essential for dopamine-induced apoptosis in PC12 cells.

The etiology of Parkinson's disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF-kappaB) is activated by oxidative stress, we studied the involvement of NF-kappaB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1-0.5 m M) treatment increased the phosphorylation of the IkappaB protein, the inhibitory subunit of NF-kappaB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF-kappaB-p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine-induced NF-kappaB activation was also evidenced by electromobility shift assay using radioactive labeled NF-kappaB consensus DNA sequence. Cell-permeable NF-kappaB inhibitor SN-50 rescued the cells from dopamine-induced apoptosis and showed the importance of NF-kappaB activation to the induction of apoptosis. Furthermore, flow cytometry assay demonstrated a higher level of translocated NF-kappaB-p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our findings suggest that NF-kappaB activation is essential to dopamine-induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinson's disease.

Increased oxidative stress in ataxia telangiectasia evidenced by alterations in redox state of brains from Atm-deficient mice.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype and suffer primarily from progressive ataxia caused by degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical features of the human disease. We previously hypothesized that some aspects of A-T, such as the preferential loss of certain neurons, could result from a continuous state of increased oxidative stress (G. Rotman and Y. Shiloh, Cancer Surv., 29: 285-304, 1997; G. Rotman and Y. Shiloh, BioEssays, 19: 911-917, 1997). The present work tests this hypothesis by analyzing markers of redox state in brains of Atm-deficient mice. We found alterations in the levels of thiol-containing compounds in Atm (-/-) brains, as well as significant changes in the activities of thioredoxin, catalase, and manganese superoxide dismutase in Atm (-/-) cerebella. These changes are indicative of increased levels of reactive oxygen species, which are seen primarily in the cerebellum of Atm-deficient mice. Our findings support the hypothesis that the absence of functional ATM results in oxidative stress, which may be an important cause of the degeneration of cerebellar neurons in A-T.

Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by activating NF-kappa B via Ref-1.

The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites. This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis. E. coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active site, penetrates into cerebellar granule neurons and exerts its activity via NF-kappaB activation. Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity. Although DA significantly reduced NF-kappaB binding activity, Grx2 could stimulate the binding of NF-kappaB to DNA by: (i) translocating NF-kappaB from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-kappaBalpha, and (ii) activating the binding of pre existing nuclear NF-kappaB. The DNA binding activity of NF-kappaB itself was essential for neuronal survival. Overexpression of I-kappaB dominant negative gene (I-kappaB-DeltaN) in granule neurons significantly reduced their viability, irrespective of the presence of Grx2. Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate NF-kappaB binding activity. These results show that Grx2 exerts its anti apoptotic activity through the activation of Ref-1, which then activates NF-kappaB.

Identification and evaluation of mental retardation.

Mental retardation in young children is often missed by clinicians. The condition is present in 2 to 3 percent of the population, either as an isolated finding or as part of a syndrome or broader disorder. Causes of mental retardation are numerous and include genetic and environmental factors. In at least 30 to 50 percent of cases, physicians are unable to determine etiology despite thorough evaluation. Diagnosis is highly dependent on a comprehensive personal and family medical history, a complete physical examination and a careful developmental assessment of the child. These will guide appropriate evaluations and referrals to provide genetic counseling, resources for the family and early intervention programs for the child. The family physician is encouraged to continue regular follow-up visits with the child to facilitate a smooth transition to adolescence and young adulthood.

The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity.

Parkinson's disease (PD) is a progressive neurological disorder caused by rather selective degeneration of the dopaminergic (DA) neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral neuronal loss is still enigmatic and treatment is basically symptomatic. The current major hypothesis suggests that nigral neuronal death in PD is due to excessive oxidative stress generated by auto- and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin and presence of high concentrations of iron. We have found that DA toxicity is mediated through its oxidative metabolites. Whereas thiol-containing antioxidants provided marked protection against DA toxicity, ascorbic acid accelerated DA-induced death. Using the differential display approach, we sought to isolate and characterize genes whose expression is altered in response to DA toxicity. We found an upregulation of the collapsin response mediator protein (CRM) and TCP-1delta in sympathetic neurons, which undergo dopamine-induced apoptosis. The isolation of these genes led us to examine the expression and activity of CRM and TCP-1delta related genes. Indeed, we found a significant induction of mRNAs of the secreted collapsin-1 and the mitochondrial stress protein HSP60. Antibodies directed against collapsin-1 provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. In a parallel study, using antisense technology, we found that inhibition of TCP-1delta expression significantly reduced DA-induced neuronal death. These findings suggest a functional role for collapsin-1 and TCP-1delta as positive mediators of DA-induced neuronal apoptosis.

The involvement of p53 in dopamine-induced apoptosis of cerebellar granule neurons and leukemic cells overexpressing p53.

1. The pathogenesis of the selective degeneration of the dopaminergic neurons in Parkinson's disease is still enigmatic. Recently we have shown that dopamine can induce apoptosis in postmitotic neuronal cells, as well as in other cellular systems, thus suggesting a role for this endogenous neurotransmitter and associated oxidative stress in the neuronal death process. 2. Dopamine has been shown to be capable of inducing DNA damage through its oxidative metabolites. p53 is a transcription factor that has a major role in determining cell fate in response to DNA damage. We therefore examined the possible correlation between dopamine-triggered apoptosis, DNA damage and levels of total phosphorylated p53 protein in cultured mouse cerebellar granule neurons. 3. Marked DNA damage and apoptotic nuclear condensation and fragmentation were detected within several hours of exposure to dopamine. An associated marked threefold increase in p53 phosphorylation was observed within this time window. Using a temperature-sensitive p53 activation system in leukemia LTR6 cells, were found that p53 inactivation dramatically attenuated dopamine toxicity. 4. We therefore conclude that DNA damage and p53 activation may have a role in mediating dopamine-induced apoptosis. Modulation of the p53 system may therefore have a protective role against the toxicity of this endogenous neurotransmitter and associated oxidative stress.

Survival and subsequent outcome to five years of age for infants with birth weights less than 801 grams born from 1983 to 1989.

OBJECTIVE: To determine the neurodevelopmental status for 5-year-old survivors with birth weights (BW) <801 gm born in the 1980s and to assess differences in outcome for those born from 1986 to 1989 compared with those born from 1983 to 1985. STUDY DESIGN: This investigation was a longitudinal follow-up of all infants with BW from 450 to 800 gm born at one of two hospitals in Kansas City, Mo., between January 1983 and December 1989. Medical records were reviewed at discharge to determine obstetric and neonatal interventions and complications. Neurodevelopmental outcome was assessed by a multidisciplinary evaluation at 60 +/- 3 months old. Multiple logistic regression analyses were used to determine factors statistically associated with survival and long-term outcome. RESULTS: The percentage of infants surviving to hospital discharge was not different between periods. From 1986 to 1989, there were more infants with BW <601 gm and <26 weeks' gestational age. Survival was greater for gestational age categories > or = 24 weeks. BW, gestational age, female gender (p < 0.0001), and birth era (p < 0.01) were each independently significantly associated with improved survival. At 5 years of age, there were no significant outcome differences between birth era cohorts. Overall, approximately 21% of infants had severe disabilities. Long-term outcome was significantly influenced by the occurrence of intracranial hemorrhage and socioeconomic status. CONCLUSION: For infants with BW <801 gm, survival of infants > or = 24 weeks' gestational age was greater in 1986 to 1989 compared with 1983 to 1985, although the 5-year neurodevelopmental outcome was not different between groups. The significant impact of social risk status on outcome suggests that factors outside the intensive care nursery must be addressed to improve long-term outcome.

High-dose phenobarbital therapy in term newborn infants with severe perinatal asphyxia: a randomized, prospective study with three-year follow-up.

OBJECTIVE: To determine whether 40 mg/kg phenobarbital given to term infants with severe asphyxia would result in a lower incidence of seizures in the newborn period and an improved neurologic outcome. METHODS: We conducted a randomized, controlled, prospective study. Entry criteria included (1) an initial arterial pH less than or equal to 7.0 with a base deficit 15 mEq/L or more, (2) Apgar score less than or equal to 3 at 5 minutes of age, or (3) failure to initiate spontaneous respiration by 10 minutes of age. Sample size was calculated to detect a 50% reduction in the incidence of neonatal seizures. RESULTS: No differences were present between treatment and control groups with respect to severity of asphyxia assessed by initial arterial pH, base excess, cerebrospinal fluid lactate dehydrogenase concentration or detection of CSF creatine kinase of its BB isoenzyme. Seizures occurred in 9 of 15 infants in the treatment group and 14 of 16 infants in the control group (p = 0.11). No adverse effects were observed from phenobarbital on heart rate, respiratory rate, blood pressure, or arterial blood gas values. Three-year follow-up revealed normal outcome in 11 of 15 infants in the treatment group and 3 of 16 in the control group (p = 0.003). CONCLUSION: Phenobarbital, when administered in a dose of 40 mg/kg intravenously over 1 hour in term, severely asphyxiated newborn infants appeared to be safe and was associated with a 27% reduction in the incidence of seizures and a significant improvement in neurologic outcome at 3 years of age.

Biochemical and temporal analysis of events associated with apoptosis induced by lowering the extracellular potassium concentration in mouse cerebellar granule neurons.

We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K+ concentration ([K+]o), a significant part of the genomic DNA was already cleaved to high-molecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K+]o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning approximately 4 h after the removal of high [K+]o and developed to nuclear condensation 4 h later. Six hours after high [K+]o deprivation, the DNA was fragmented into oligonucleosome-sized fragments. Within 6 h after removal of the extracellular K+, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K+]o. Similar to high [K+]o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K+]o removal. An approximately 40% decrease in RNA and protein synthesis was detected by 6 h of [K+]o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome-sized fragments, and the reduction in mitochondrial activity-events that occurred within the critical time-may indicate that these processes have an important part in the mechanism that committed the neurons to die.

Selective loss of sperm bearing a compound chromosome in the Drosophila female.

The Drosophila compound entire second chromosome, C(2)EN, displays paternal transmission well below Mendelian expectations (NOVITSKI et al. 1981). Because C(2)EN stocks also show higher-than-expected rates of zygotic lethality, it was proposed that this reduced paternal inheritance might be wholly or partially due to postfertilization events. Efforts to investigate this phenomenon have been hampered because the progeny of crosses between C(2)EN-bearing individuals and those with normal karyotypes die during embryogenesis. We have circumvented this obstacle by employing fluorescence in situ hybridization to directly karyotype early embryos from crosses involving C(2)EN-bearing individuals. This analysis reveals that the distortion in paternal transmission is established before fertilization. Moreover, measurement of the sperm ratios within both the male and female reproductive organs demonstrates that C(2)EN-bearing sperm are selectively lost after sperm transfer to the female and before storage of sperm in the seminal receptacles and spermathecae. Our results are consistent with a model of meiotic drive in which aberrations occurring early in meiosis lead ultimately to sperm dysfunction.

Live analysis of free centrosomes in normal and aphidicolin-treated Drosophila embryos.

In a number of embryonic systems, centrosomes that have lost their association with the nuclear envelope and spindle maintain their ability to duplicate and induce astral microtubules. To identify additional activities of free centrosomes, we monitored astral microtubule dynamics by injecting living syncytial Drosophila embryos with fluorescently labeled tubulin. Our recordings follow multiple rounds of free centrosome duplication and separation during the cortical division. The rate and distance of free sister centrosome separation corresponds well with the initial phase of associated centrosome separation. However, the later phase of separation observed for centrosomes associated with a spindle (anaphase B) does not occur. Free centrosome separation regularly occurs on a plane parallel to the plasma membrane. While previous work demonstrated that centrosomes influence cytoskeletal dynamics, this observation suggests that the cortical cytoskeleton regulates the orientation of centrosome separation. Although free centrosomes do not form spindles, they display relatively normal cell cycle-dependent modulations of their astral microtubules. In addition, free centrosome duplication, separation, and modulation of microtubule dynamics often occur in synchrony with neighboring associated centrosomes. These observations suggest that free centrosomes respond normally to local nuclear division signals. Disruption of the cortical nuclear divisions with aphidicolin supports this conclusion; large numbers of abnormal nuclei recede into the interior while their centrosomes remain on the cortex. Following individual free centrosomes through multiple focal planes for 45 min after the injection of aphidicolin reveals that they do not undergo normal modulation of their astral dynamics nor do they undergo multiple rounds of duplication and separation. We conclude that in the absence of normally dividing cortical nuclei many centrosome activities are disrupted and centrosome duplication is extensively delayed. This indicates the presence of a feedback mechanism that creates a dependency relationship between the cortical nuclear cycles and the centrosome cycles.

Seasonal respiratory viral infections. Impact on infants with chronic lung disease following discharge from the neonatal intensive care unit.

OBJECTIVE: To determine the frequency and severity of acute respiratory infections in infants with bronchopulmonary dysplasia following discharge from the neonatal intensive care unit. DESIGN: A prospective cohort study of 30 oxygen-dependent children who were younger than 2 years (mean age, 9.8 months; range, 3 to 24 months) were studied from September 1990 through April 1991. MEASUREMENTS/RESULTS: During the study, 101 (90.2%) of 112 visits for illness were prompted by new or worsening respiratory symptoms. Diagnoses included upper respiratory tract infection (30.4%), otitis media (26.0%), pneumonia (11.1%), acute exacerbation of bronchopulmonary dysplasia (10.4%), reactive airway disease (9.6%), and bronchiolitis (5.9%). Among these children, an increase in the fraction of inspired oxygen was necessary during 43% of visits. Ten children were hospitalized on 25 occasions for a mean of 37.6 hospital days per child (range, 1 to 107 days), and mean length of stay for each hospitalization was 15 days (median, 6 days). Five children were admitted to the pediatric intensive care unit. Respiratory viruses isolated included respiratory syncytial virus (n = 7), parainfluenza 3 virus (n = 3), and adenovirus (n = 2). No isolates of influenza A or B were detected. Anthropometrics at study entry and study end were converted to z scores as descriptors of weight for age, height for age, and weight for height. Growth improved during the 8 months of the study; however, overall, the children were leaner at study end than at study entry. CONCLUSIONS: In children with bronchopulmonary dysplasia, respiratory viral infections led to significant morbidity, which included long and frequent hospitalizations during the peak of the respiratory viral season. Although weight and linear growth increased throughout the study, patients were leaner at study conclusion than at study entry.

Growth patterns for infants weighing less than 801 grams at birth to 3 years of age.

Anthropometric measurements of infants weighing < 801 gm were monitored to evaluate growth patterns from birth to 36 months chronologic age, using both standard growth percentiles and Z score descriptors. Mean gestational age was 25.8 weeks, and birth weight was 708.4 gm. The infants had growth delay at hospital discharge with some catch-up growth occurring in the first year. Discrete changes in body growth using the Z score as a descriptor showed movement from a negative SD at 12 months toward the reference mean of zero at 36 months. However, mean length remained < 5th percentile, and mean weight was at approximately the 10th percentile for chronologic age. At 36 months 45% of infants had a head circumference smaller than the 5th percentile for chronologic age. Birth weight < 701 gm was a significant predictor of smaller occipital-frontal circumference at 3 years (p = 0.03). Interruption of intrauterine growth after extremely premature birth appears to have long-term effects on growth outcome much like prenatal growth retardation.

Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo.

We employ fluorescently labeled dextran beads to follow cycles of nuclear division and nuclear envelope breakdown in living Drosophila embryos. When injected into syncytial Drosophila embryos, 3000 mol wt fluorescently labeled dextran beads concentrate in the interphase nucleus. Through confocal microscopy, undisrupted multiple cycles of nuclear division are readily followed. In contrast, 40,000 mol wt fluorescently labeled dextran beads concentrate in the cytoplasm and enter the nucleus only after nuclear envelope breakdown during prophase. Once the nuclear envelope reforms during telophase, these large dextran beads are again excluded from the nuclei. The complementary behavior of the large and small dextran beads makes them applicable to a broad range of cellular studies. We employ the 40,000 mol wt fluorescein-labeled beads, in combination with rhodamine-labeled histones, to demonstrate that nuclear envelope breakdown occurs about 3.5-4.0 minutes prior to the initiation of anaphase.

Delays in anaphase initiation occur in individual nuclei of the syncytial Drosophila embryo.

The syncytial divisions of the Drosophila melanogaster embryo lack some of the well established cell-cycle checkpoints. It has been suggested that without these checkpoints the divisions would display a reduced fidelity. To test this idea, we examined division error frequencies in individuals bearing an abnormally long and rearranged second chromosome, designated C(2)EN. Relative to a normal chromosome, this chromosome imposes additional structural demands on the mitotic apparatus in both the early syncytial embryonic divisions and the later somatic divisions. We demonstrate that the C(2)EN chromosome does not increase the error frequency of the late larva neuroblast divisions. However, in the syncytial embryonic nuclear divisions, the C(2)EN chromosome produces a 10-fold increase in division errors relative to embryos with a normal karyotype. During late anaphase of the neuroblast divisions, the sister C(2)EN chromosomes cleanly separate from one another. In contrast, during late anaphase of the syncytial divisions in C(2)EN-bearing nuclei, large amounts of chromatin often lag on the metaphase plate. Live analysis of C(2)EN-bearing embryos demonstrates that individual nuclei in the syncytial population of dividing nuclei often delay in their initiation of anaphase. These delays frequently lead to division errors. Eventually the products of the nuclei delayed in anaphase sink inward and are removed from the dividing population of syncytial nuclei. These results suggest that the Drosophila embryo may be equipped with mechanisms that monitor the fidelity of the syncytial nuclear divisions. Unlike checkpoints that rely on cell cycle delays to identify and correct division errors, these embryonic mechanisms rely on cell cycle delays to identify and discard the products of division errors.

Systemic hypertension in infants with severe bronchopulmonary dysplasia: associated clinical factors.

The clinical course of 87 infants with bronchopulmonary dysplasia (BPD) on home oxygen therapy was reviewed to determine the occurrence of systemic hypertension (HTN) and to evaluate associated clinical features. Eleven of 87 (13%) infants developed systemic HTN either in the neonatal intensive care unit or following discharge. Clinical features that distinguished the hypertensive from the normotensive group were as follows: greater use of bronchodilators, 91% vs 37% (p < 0.001), and diuretics, 91% vs 55% (p < 0.05), longer duration of home oxygen therapy 21.6 +/- 9.9 vs 9.2 +/- 5.8 months (p < 0.05), and greater mortality, 36% vs 1% (p < 0.001). The course of systemic HTN in the surviving patients (7 of 11) was benign and resolved in all patients prior to weaning from home oxygen therapy. Systemic HTN is frequently present in infants with severe BPD and appears to be related to the clinical severity of lung disease.

Kidney function in very low birth weight infants with furosemide-related renal calcifications at ages 1 to 2 years.

To determine whether long-term renal sequelae follow the use of furosemide in preterm infants, we evaluated renal function in 27 former very low birth weight infants (less than 1500 gm) at 1 to 2 years of age. Patients were classified into three groups on the basis of status at the time of discharge from the hospital: group 1 (n = 7) had no furosemide treatment or renal calcifications, group 2 (n = 10) had furosemide therapy but no calcifications, and group 3 (n = 10) had furosemide therapy with renal calcifications. Renal ultrasonography at the time of the study demonstrated resolution of the calcifications in six patients in group 3. No differences in renal function were observed between groups 1 and 2. Creatinine clearance (mean +/- SEM) in group 3 (83.6 +/- 7.8 ml/min per 1.73 m2) was significantly lower than clearance in groups 1 and 2 (103.2 +/- 6.5 and 109.1 +/- 5.1, respectively; p less than 0.05). Children in group 3 had significantly higher urinary calcium/creatinine ratios and fractional excretion of sodium and lower tubular reabsorption of phosphate than children in the two other groups had. Urine-blood difference in carbon dioxide tension after oral acetazolamide load, which indicates the ability of the distal tubule to secrete hydrogen ions, was 8.4 +/- 3.4 mm Hg in group 3, significantly lower than values in groups 1 and 2 (22.6 +/- 3.1 and 28.0 +/- 4.3 mm Hg, respectively, p less than 0.05). Within group 3 the four children with persistent renal calcifications had significantly lower urine-blood carbon dioxide tension differences than did those with resolution of calcifications (p = 0.02). We conclude that furosemide-related renal calcifications in very low birth weight infants may lead to glomerular and tubular dysfunction; further long-term follow-up of this population is recommended.