Beta amyloid gene duplication in Alzheimer's disease and karyotypically normal Down syndrome.

With the recently cloned complementary DNA probe, lambda Am4 for the chromosome 21 gene encoding brain amyloid polypeptide (beta amyloid protein) of Alzheimer's disease, leukocyte DNA from three patients with sporadic Alzheimer's disease and two patients with karyotypically normal Down syndrome was found to contain three copies of this gene. Because a small region of chromosome 21 containing the ets-2 gene is duplicated in patients with Alzheimer's disease, as well as in karyotypically normal Down syndrome, duplication of a subsection of the critical segment of chromosome 21 that is duplicated in Down syndrome may be the genetic defect in Alzheimer's disease.

A critical role for the glial-derived neuromodulator D-serine in the age-related deficits of cellular mechanisms of learning and memory.

Age-associated deficits in learning and memory are closely correlated with impairments of synaptic plasticity. Analysis of N-methyl-D-aspartate receptor (NMDAr)-dependent long-term potentiation (LTP) in CA1 hippocampal slices indicates that the glial-derived neuromodulator D-serine is required for the induction of synaptic plasticity. During aging, the content of D-serine and the expression of its synthesizing enzyme serine racemase are significantly decreased in the hippocampus. Impaired LTP and NMDAr-mediated synaptic potentials in old rats are rescued by exogenous D-serine. These results highlight the critical role of glial cells and presumably astrocytes, through the availability of D-serine, in the deficits of synaptic mechanisms of learning and memory that occur in the course of aging.

Selenium-dependent and non-selenium-dependent glutathione peroxidases in human tissue extracts.

A method for the assessment of both selenium-dependent and non-selenium-dependent glutathione peroxidases on crude tissue extracts in human is described. The enzyme activity is measured by the coupled assay system in which oxidation of reduced glutathione (GSH) is coupled to NADPH oxidation catalyzed by glutathione reductase. Total glutathione peroxidase activity is measured with cumene hydroperoxide as substrate. Selenium-dependent glutathione peroxidase is measured with tert-butyl hydroperoxide. This substrate is preferable to H2O2 which gives too high blank values compared to the assay values. The difference between total glutathione peroxidase and selenium-dependent glutathione peroxidase activities represents the non-selenium-dependent glutathione peroxidase activity. Studies of substrate specificity of the two glutathione peroxidases separated by gel filtration as well as linearity and recovery studies are presented. For a given tissue, the relative amounts of the two glutathione peroxidases given by our assay are identical to those estimated by quantifying the elution peaks after gel filtration. Based on the percentages of the two glutathione peroxidases, human tissues can be classified in four groups: (1) the non-selenium-dependent glutathione peroxidase is predominant in liver, in renal cortex and skeletal muscle; (2) non-selenium-dependent and selenium-dependent glutathione peroxidases are in equal amounts in renal medulla; (3) the selenium-dependent glutathione peroxidase is predominant in adrenal glands and platelets; (4) the selenium-dependent glutathione peroxidase represents 100% of the glutathione peroxidase activity in the other organs. The heart and the brain are of special interest in this group because of the physiological role and the regulation of the selenoenzyme.

Expression of transfected human CuZn superoxide dismutase gene in mouse L cells and NS20Y neuroblastoma cells induces enhancement of glutathione peroxidase activity.

The human CuZn superoxide dismutase (superoxide dismutase 1) a key enzyme in the metabolism of oxygen free-radicals, is encoded by a gene located on chromosome 21 in the region 21 q 22.1 known to be involved in Down's syndrome. A gene dosage effect for this enzyme has been reported in trisomy 21. To assess the biological consequences of superoxide dismutase 1 overproduction within cells, the human superoxide dismutase 1 gene and a human superoxide dismutase 1 cDNA were introduced into mouse L cells and NS20Y neuroblastoma cells. Both cell types expressed elevated levels (up to 3-fold) of enzymatically active human superoxide dismutase 1. These human superoxide dismutase 1 overproducers, especially neuronal cell lines, showed an increased activity in the selenodependent glutathione peroxidase. These data are consistent with the possibility that gene dosage of superoxide dismutase 1 contributes to oxygen metabolism modifications previously described in Down's syndrome.

Developmentally regulated expression of mtprd, the murine ortholog of tprd, a gene from the Down syndrome chromosomal region 1.

The gene tprd, which contains three tetratricopeptide domains, has been recently localized in the Down syndrome (DS) chromosomal region 1. We have cloned a cDNA encoding part of the murine ortholog of tprd and used it to characterize the expression pattern of this gene during development and at the adult stage. At E8.5 the expression is uniform. In the later stages of embryogenesis, although expression remains ubiquitous, a pattern of tissues with particularly high expression develops: the strong expression is restricted to non proliferating zones of the nervous system such as the external layer of the cortex, the spinal cord, the cranial and root ganglia and the nerves. In the brain of adult mouse the strongest signals are observed in layers II-III and V-VI of the cortex, in the hippocampus and in the cerebellum, which correspond to the abnormal brain regions seen in DS patients.

Regional and cellular specificity of the expression of TPRD, the tetratricopeptide Down syndrome gene, during human embryonic development.

The TPRD gene (tetratricopeptide (TPR) containing Down syndrome gene) is one of the candidate genes in the Down syndrome chromosomal region-1. Duplication of this gene may be the cause of major phenotypic features of Down syndrome. Here we show that the TPRD expression is developmentally regulated during human embryogenesis. At the earliest stages of development (Carnegie 8-12) TPRD expression is ubiquitous. At later developmental stages (Carnegie stages 14, 16 and 18), it becomes restricted to the nervous system, as is the case for the mtprd gene during mouse development. We extended our analysis of TPRD expression during fetal development of the human nervous system (13, 22 and 24 weeks). A new oblique illumination technique was used to compare signal intensity and cell density. Some regions of the nervous system such as the external cortical layers of the brain, and the inner neuroblastic layer of the eye, strongly express the TPRD gene.

Molecular mapping of twenty-four features of Down syndrome on chromosome 21.

To determine which regions of chromosome 21 are involved in the pathogenesis of specific features of Down syndrome, we analysed, phenotypically and molecularly, 10 patients with partial trisomy 21. Six minimal regions for 24 features were defined by genotype-phenotype correlations. Nineteen of these features could be assigned to just 2 regions: short stature, joint hyperlaxity, hypotonia, major contribution to mental retardation and 9 anomalies of the face, hand and foot to the region D21S55, or Down syndrome chromosome region (DCR), located on q22.2 or very proximal q22.3, and spanning 0.4-3 Mb; 6 facial and dermatoglyphic anomalies to the region D21S55-MX1, including the DCR and spanning a maximum of 6 Mb on q22.2 and part of q22.3. Thus, the complex phenotype that constitutes Down syndrome may in large part simply result from the overdosage of only one or a few genes within the DCR and/or region D21S55-MX1.

Mapping the Down syndrome chromosome region. Establishment of a YAC contig spanning 1.2 megabases.

The triplication of a region of chromosome 21 around D21S55 in 21q22.2-22.3 has been involved in the main features of Down syndrome including mental retardation (Down syndrome chromosome region: DCR). To improve the physical map of this region, we screened yeast artificial chromosome (YAC) libraries with ETS2 and ERG sequences. Five selected clones were analyzed by AluPCR, pulsed-field gel electrophoresis, and in situ hybridization. A 1.2-Mg contig, encompassing the protooncogenes ETS2 and ERG, was identified, its restriction map established and compared to the genomic map. ERG is distal to D21S55 and proximal to ETS2. ERG and ETS2 genes are 400 kb apart and in opposite orientations. The contig contains the distal boundary and part of the DCR. Three putative HTF islands were identified.

Preferential expression of superoxide dismutase messenger RNA in melanized neurons in human mesencephalon.

The copper-zinc-dependent superoxide dismutase messenger RNA expression was studied at cellular level by in situ hybridization, using a 35S-labelled complementary DNA probe homologous to human copper-zinc-dependent superoxide dismutase messenger RNA, in the dopaminergic neuron-containing areas of the human mesencephalon (the substantia nigra pars compacta, ventral tegmental area, central gray substance and peri- and retrorubral region corresponding to catecholaminergic cell group A8). The autoradiographic labelling signal was localized in neurons. No detectable hybridization signal could be found in the glial cells. Copper-zinc-dependent superoxide dismutase messenger RNA was detected in melanin-containing neurons as well as in non-melanized neurons. Quantification at cellular level, taking the autoradiographic silver grain density as an index of the abundance of copper-zinc-dependent superoxide dismutase messenger RNA, indicated that hybridization level was higher in the melanized than in the non-melanized neurons within a region. Among melanized neurons, cellular copper-zinc-dependent superoxide dismutase messenger RNA content was lowest in the neurons of the substantia nigra. No significant difference in levels of transcripts was evidenced between the groups of non-melanized neurons. The data suggest that the abundance of copper-zinc-dependent superoxide dismutase messenger RNA is higher in the mesencephalic neurons containing neuromelanin compared to other neurons. Thus, the melanized neurons have a particular defence system against oxygen toxicity, which may represent a basis for their preferential vulnerability to Parkinson's disease.

Accurate evaluation of the sizes of DNA fragments (from 30 to 4700 kb) in pulse field gel electrophoresis.

Construction of long-range genomic maps by pulse field gel electrophoresis requires optimum resolution of large DNA fragments. Using the transverse-alternating field electrophoresis system, we describe a method to accurately evaluate the sizes of fragments generated by rare-cutter digestions within the 30-4700-kb range. A protocol generating large (greater than 1000 kb) molecules by partial digestion is also reported.

H2O2 production, modification of the glutathione status and methemoglobin formation in red blood cells exposed to diethyldithiocarbamate in vitro.

Human red blood cells treated with the CuZn superoxide dismutase inhibitor diethyldithiocarbamate (DDC) undergo metabolic modifications in addition to the superoxide dismutase inhibition: oxidation of the reduced glutathione (GSH) to oxidized glutathione (GSSG), methemoglobin formation, and increased hexose monophosphate shunt activity were observed. The magnitudes of these changes are dependent on the DDC concentration. Under nitrogen, only superoxide dismutase inhibition occurs. After removal of the GSH with N-ethylmaleimide, production of H2O2 can be detected by measuring the red cell catalase inhibition in the presence of 3-amino-1,2,4-triazole. H2O2 production is not altered by conversion of oxyhemoglobin to methemoglobin by sodium nitrite prior to incubation. GSH oxidation and methemoglobin formation are stopped when DDC is eliminated from the incubation medium after completion of the superoxide dismutase inhibition. These data indicate that methemoglobin formation and modification of the GSH status in red cells treated by DDC are not a direct consequence of the CuZn superoxide dismutase inhibition but are due rather to a DDC-dependent production of H2O2.

Cellular clones and transgenic mice overexpressing copper-zinc superoxide dismutase: models for the study of free radical metabolism and aging.

Down's Syndrome (DS), the most frequent of congenital birth defects, results from the trisomy of the chromosome numbered 21 in all cells of affected patients. This disease is characterized by developmental anomalies, mental retardation and features of rapid aging, particularly in the brain where the occurrence of Alzheimer's disease (AD) is observed in all trisomy 21 patients over the age of 35. Elucidation of the biological mechanisms leading to brain aging in DS might provide new insight into the understanding of brain aging and AD in normal people. Copper-zinc superoxide dismutase (CuZnSOD) is one of the genes encoded by chromosome 21. As a consequence of gene dosage excess, CuZnSOD activity and protein are increased by 50% in all DS tissues. The level of CuZnSOD protein and mRNA is particularly high in hippocampal pyramidal neurons susceptible to degenerative processes in AD and in dopaminergic melanized-neurons vulnerable in Parkinson's disease. Increased CuZnSOD activity in these age-related neurodegenerative disorders might result in H2O2 overproduction and subsequently promote peroxidative damages within cells. Increase of seleno-dependent glutathione peroxidase (Se-GPx) in DS cells supports this concept. In order to test this hypothesis, cell and animal models of CuZnSOD overexpression have been designed. In cells transfected with the human CuZnSOD gene, and increased Se-GPx activity is observed, a situation which mimics DS. In mice transgenic for the human CuZnSOD, the expression pattern of the transgene in the brain is similar to that in humans, and we can observe an increased peroxidation in this tissue. These data, like others in the literature, support the hypothesis that excess CuZnSOD induces an imbalance in the regulation of oxygen-derived free radical production which might result in peroxidative brain damage and possibly contribute to accelerated aging and age-related neuropathology.

Neuronal-specific expression of human copper-zinc superoxide dismutase gene in transgenic mice: animal model of gene dosage effects in Down's syndrome.

It has been suggested that copper-zinc superoxide dismutase (CuZn SOD) increment, by accelerating hydrogen peroxide formation, might promote oxidative damage within trisomy 21 cells and might be involved in the various neurobiological abnormalities found in Down's syndrome such as premature aging and Alzheimer-type neurological lesions. In order to test this hypothesis, we have developed strains of transgenic mice carrying the human CuZn SOD gene. The human transgene expression resulted in increased CuZn SOD activity predominantly in the brain (1.93 fold). Immunohistochemical and in situ hybridization analysis of brain sections revealed that human CuZn SOD protein and mRNA was preferentially expressed in neurons, particularly in pyramidal cells of Ammon's horn and granule cells of gyrus dentate. The amount of thiobarbituric acid (TBA)-reactive material was significantly higher in transgenic brains compared to controls, strongly suggesting an increased level of peroxidation in vivo. These results support the notion that CuZn SOD gene dosage effect could play a role in the pathogenesis of rapid aging features in the brain of Down's syndrome patients.

Overexpression of mSim2 gene in the zona limitans of the diencephalon of segmental trisomy 16 Ts1Cje fetuses, a mouse model for trisomy 21: a novel whole-mount based RNA hybridization study.

Trisomy 21 (Down syndrome) is the most common chromosomal abnormality associated with mental retardation in humans. Sim2, a human homologue of Drosophila sim gene, which acts as a master regulator of the early development of the fly central nervous system midline, is located on chromosome 21, in the Down syndrome critical region, and might therefore be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down syndrome. We report here the detailed expression pattern of murine mSim2 gene in Ts1Cje mice fetuses, a segmental trisomy 16 mouse model for trisomy 21, and its overexpression in the zona limitans of the diencephalon using a new quantitative method based on the whole-mount RNA hybridization technique.

Preferential localization of copper zinc superoxide dismutase in the vulnerable cortical neurons in Alzheimer's disease.

The distribution of cells containing CuZn superoxide dismutase (CuZn SOD) was determined in hippocampi and associative cortex from normal and Alzheimer's individuals by using antisera against native and denatured CuZn SOD proteins. Immunostaining was intense in large pyramidal neurons, moderate in hippocampal granule cells and very weak in other cells. In the hippocampus of an Alzheimer's patient, successive immunostaining of the same tissue section by anti CuZn SOD and anti paired helical filaments antisera show that both normal and degenerating cells are labelled by the anti CuZn SOD antiserum. Thus, large pyramidal neurons which are potentially susceptible to degenerative processes in AD have the property to contain higher amounts of CuZn SOD than other brain cells.

Reduced protein kinase C activity in sporadic Alzheimer's disease fibroblasts.

A decrease in the protein kinase C immunoreactivity and an altered protein phosphorylation have been reported in patients with Alzheimer's disease, but discordant results have been obtained from determinations of protein kinase C activity. By assaying the calcium- and phospholipid-dependent phosphorylation of a lysine-rich histone after detergent extraction, we have determined the total protein kinase C activity in fibroblasts from patients with sporadic Alzheimer's disease, age-matched controls and young subjects. The activity was not significantly different between young and aged controls, whereas it was significantly lower (0.70 +/- 0.12 vs 1.16 +/- 0.23 nmol/min/mg protein, P less than 0.01) in the patients. The total amount of protein kinase C estimated from the binding of phorbol dibutyrate to intact cells was also significantly lower (1.70 +/- 0.41 vs 2.48 +/- 0.54 pmol/mg protein, P less than 0.01). This decrease in protein kinase C activity suggests that abnormal protein phosphorylation might play a role in the pathogenesis of the disease.

Localization of copper-zinc superoxide dismutase mRNA in human hippocampus by in situ hybridization.

The cellular localization of copper-zinc superoxide dismutase (CuZn SOD) mRNA was determined in the human hippocampus by in situ hybridization with a 35S-labelled DNA probe complementary to human CuZn SOD mRNA. A positive hybridization signal was detected in pyramidal cell layers CA1-CA4 of Ammon's horn (CA), pyramidal cells of subiculum and in the granule cells of the dentate gyrus. The fact that CuZn SOD gene expression is important in neurones which are preferentially vulnerable in neurodegenerative processes such as Alzheimer's disease, suggests a role played by oxygen free radicals in the mechanism of nerve cell death.

Hippocampal mossy fiber changes in mice transgenic for the human copper-zinc superoxide dismutase gene.

The copper-zinc superoxide dismutase (SOD-1) gene, located on chromosome 21 and triplicated in Down's syndrome (DS), is suspected to be involved in the neuropathology observed in Alzheimer's disease (AD), DS and physiological aging. In order to explore the effect of an overproduction of SOD-1 in the mouse hippocampus, we investigated the Timm-stained mossy fiber (MF) innervation in the hippocampus of transgenic mice for the human SOD-1 gene (hSOD-1 mice). The results showed a decrease of the MF projection area in the hSOD-1 mice overexpressing the SOD-1 protein. These findings suggest that free radicals could play a role in this particular synaptic loss.

Glutathione-S-transferase of human red blood cells; assay, values in normal subjects and in two pathological circumstances: hyperbilirubinemia and impaired renal function.

An assay for human erythrocyte glutathione-S-transferase is described. The procedure is both sensitive and reproducible; sampling and storage conditions are also investigated. Reference values are given for normal neonates, children and adults. The normal red cell glutathione-S-transferase activity decreases significantly during the first weeks of life and remains constant afterwards. A slight but significant increase is observed over 75 years. Sex difference has no influence on enzyme activity. A significant increase (up to fourfold) in red cell glutathione-S-transferase activity is noted in newborns with hyperbilirubinemia exceeding 135 mumol/l. Red cell glutathione-S-transferase is also significantly increased in hemodialysed subjects suffering from renal dysfunction.

Selenium, zinc and copper in Down's syndrome (trisomy 21): blood levels and relations with glutathione peroxidase and superoxide dismutase.

Increased superoxide dismutase and glutathione peroxidase activities have been reported in erythrocytes of subjects with Down's syndrome. Since these enzymes contain specific trace-elements as essential components, we have determined copper, zinc and selenium levels in plasma and erythrocytes of 29 trisomy 21 patients compared with 32 age-matched controls and examined the relations with the enzymes' activities. In plasma, mean zinc and copper levels were normal, but selenium was found to be significantly decreased (p less than 0.001). In red cells, the increase of activity of the selenoenzyme glutathione peroxidase (p less than 0.001) was not accompanied by an increase of erythrocyte selenium, but a significant correlation was found between these two values (r = 0.67, p less than 0.001). Zinc and copper levels in red cells were significantly higher than normal (p less than 0.001) and this increase could be partly explained by the increased activity of the copper and zinc containing enzyme superoxide dismutase (p less than 0.001). Low plasma selenium and the strong relation between erythrocyte selenium and glutathione peroxidase activity we found in Down's syndrome should stimulate interest in a more detailed investigation of selenium status and metabolism of these patients.