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(The FASEB Journal. 2004;18:62-69.)
© 2004 FASEB

DSCR1(Adapt78) modulates expression of SOD1

GENNADY ERMAK*, CHRIS CHEADLE{dagger}, KEVIN G. BECKER{dagger}, CATHRYN D. HARRIS* and KELVIN J. A. DAVIES*,1

* Ethel Percy Andrus Gerontology Center, and Division of Molecular & Computational Biology, The University of Southern California, Los Angeles, CA 90089-0191, USA; and
{dagger} DNA Array Unit, Research Resources Branch, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore MD 21224-6825, USA

1Correspondence: Ethel Percy Andrus Gerontology Center, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089-0191, USA. E-mail: kelvin{at}usc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DSCR1(Adapt78) is a stress responsive gene that can be induced by multiple stresses. We have previously demonstrated that acute DSCR1(Adapt78) overexpression can transiently protect cells against oxidative stress and calcium-mediated stresses, while its chronic overexpression is associated with neurofibrillary tangles, Alzheimer disease, and Down’s syndrome. It seems that a delicate balance of DSCR1(Adapt78) expression is maintained in cells, and this gene can have either protective or damaging effects, depending on both its level and duration of expression. The mechanisms by which DSCR1(Adapt78) can protect or harm cells are poorly understood. Here, we tried to identify pathways and targets affected by the DSCR1(Adapt78) gene using regulated expression of DSCR1(Adapt78) in PC-12 cells, followed by microarray analysis of mRNAs from these cells. We found that DSCR1(Adapt78) expression stimulates SOD1 (intracellular Cu,Zn superoxide dismutase) gene expression and increased sod 1 enzyme activity. Previous studies have indicated that sod 1 can either protect or damage cells, depending on its levels. Our findings suggest that sod 1 may also be involved in both the acute protective and the chronic damaging effects of DSCR1(Adapt78) expression. These data also have importance for our understanding of Down’s syndrome, Alzheimer’s disease, and other human pathologies.


Key Words: calcipressin 1 • sod 1 • oxidative stress • Down’s syndrome


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE DSCR1(Adapt78) GENE was isolated from the hamster genome as a gene induced during transient adaptation to oxidative stress (1) and simultaneously from Down’s syndrome candidate region 1 of human chromosome 21, the trisomy of which causes Down’s syndrome (2) . It has now been demonstrated that DSCR1(Adapt78) belongs to a new family of genes that bind and inhibit calcineurin (3 , 4) . The name "Calcipressin 1" has been suggested for the calcineurin inhibitory DSCR1(Adapt78) protein product.

Calcineurin is a calcium/calmodulin activated serine/threonine phosphatase (PP2B) that is an important enzyme in Ca2+-dependent eukaryotic signal transduction pathways (5) . It plays important roles in immune stimulation, and calcineurin-dependent signal transduction pathways have been extensively characterized during T cell activation (6) . Calcineurin transduction pathways are also well characterized in yeast, where the protein promotes growth in high calcium environments by dephosphorylation of the Tcn1p transcription factor (7 8 9) . Calcineurin plays a critical role in cellular responses to various extracellular signals and environmental stresses, and it is important in the regulation of apoptosis (9 10 11 12 13) , memory processes (14 15 16) , and skeletal and cardiac muscle growth and differentiation (17 , 18) . In plants calcineurin mediates salt adaptation (19) . Since the DSCR1(Adapt78) protein, "Calcipressin 1" can regulate calcineurin activity, it is likely to be involved as an antagonist in many of the above processes.

The DSCR1(Adapt78) gene consists of 7 exons, four of which (exons 1–4) can be alternatively spliced to produce a number of different mRNA isoforms (20) . These isoforms may have different expression patterns, functions, and different regulation mechanisms. For example, expression of exon 2 has been detected in fetal, but not adult human brain (2) , and calcineurin can induce expression of DSCR1(Adapt78) isoform 4 mRNA but does not appear to induce the other isoforms (21) .

DSCR1(Adapt78) was discovered as a gene associated with transient adaptation to oxidative stress and calcium stress (1) , and we have recently demonstrated that it can actually provide stress protection. However, we also observed that this gene is overexpressed in Alzheimer’s disease and Down’s syndrome, indicating that its expression can also be harmful. Therefore, we hypothesized that although acute and transient DSCR1(Adapt78) overexpression may protect against calcium-mediated stresses, its chronic overexpression can cause cell damage (22 23 24) . The mechanisms by which DSCR1(Adapt78) can protect or harm cells are still unclear. Here, using regulated expression of a DSCR1(Adapt78) transgene, followed by microarray analysis, we have tried to identify pathways and targets affected by its expression.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All chemicals and supplies were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.

Doxycycline regulated DSCR1(Adapt78) expression system
DSCR1(Adapt78) isoform 1 fragment, produced by LA RT-PCR as we described previously (23) , was inserted into a pTRE vector from Clontech Laboratories, Inc. (Palo Alto, CA). PC-12 tet-off cells from Clontech Laboratories, Inc., stably transfected with "regulator plasmid," were next transfected with the pTRE carrying DSCR1(Adapt78) fragment to finally produce a double-stable cell line in which the DSCR1(Adapt78) transgene could be turned off by doxycycline. All procedures were performed as described in the tet-off gene expression user manual from Clontech Laboratories, Inc.

PC-12 cell culture
PC-12 cells were cultivated in media containing 85% DMEM, 10% horse serum, 5% antibiotic free fetal bovine serum (Clontech Laboratories, Inc.), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin sulfate. PC-12 tet-off cells, stably transfected with "regulator plasmid," were maintained in media additionally containing 50 µg/ml G418. The double-stable cell line carrying both the "regulator plasmid" and the DSCR1(Adapt78) transgene was maintained in media additionally containing 50 µg/ml G418 and 50 µg/ml hygromycin. Cells were cultivated in a humidified 10% CO2 atmosphere at 37°C. The DSCR1(Adapt78) transgene was turned off by application of 0.02 µg/ml doxycycline to the media.

RNA isolation
Total RNA was extracted using the TRIzol reagent (Life Technologies, Inc., Rockville, MD). RNA concentration was quantified spectrophotometrically, and relative content was further confirmed on ethidium bromide-stained gels. Integrity of the RNA was estimated by agarose gel electrophoresis; only RNA samples displaying discrete 28S and 18S RNA bands were used in our experiments.

Northern hybridization
Samples containing 10 µg of total RNA were subjected to electrophoresis through 1% agarose formaldehyde gels, blotted to nylon membranes (Oncor) with HETS (CINNA/BIOTECX), and cross-linked by ultraviolet radiation. The membranes were prehybridized for 4 h and then hybridized for 15 h in Hybrizol I (Oncor) at 42°C. After hybridization, they were washed with 2xSSC plus 0.1% SDS at room temperature for 1 and 10 min, then with 0.1xSSC plus 0.1% SDS at 60°C for 10 and 30 min. The membranes were exposed, developed, and scanned using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). To rehybridize RNA gel blots, probes were removed by washing the membranes in a solution of 0.1xSSC, 0.1% SDS, and 10 mM Tris-HCl (pH 7.0) at 90°C for 10 min. To quantify the levels of DSCR1(Adapt78) mRNA, the membranes were scanned and the hybridization signal measured using ImageQuant software (Molecular Dynamics). Each signal was recalculated according to the amount of RNA actually loaded on the gels. The amount of RNA loaded was controlled in two ways. First, the gels were stained with ethidium bromide, photographed under UV light, and the amount of 28S and 18S was evaluated. Second, membranes were hybridized with a GAPDH probe. Probes containing [{alpha}-32P]dCTP-labeled DNA were prepared using the High Prime system (Boeringer Mannheim, Germany). A PCR fragment corresponding to DSCR1(Adapt78) isoform 1 was used to prepare the DSCR1(Adapt78) probe and a PCR-fragment consisting of GAPDH exons 7 and 8 was used to prepare GAPDH probes. PCR fragments were prepared as we described previously (23) .

Microarray construction and use
A 15,000 human cDNA clone set of IMAGE Consortium clones (http://image.llnl.gov) available from Research Genetics (Huntsville, AL, USA) was sorted for genes relevant to signal transduction, immune function, and neurological importance. A focused set of 1152 genes for each array type was PCR amplified and printed in duplicate at high density onto nylon membranes. The NIA immunoarray (25) and the NIA Neuroarray (26) have been described previously. cDNAs arrayed for the Signal Transduction array include receptor genes of the tyrosine kinase, tyrosine phosphate, and G-protein-coupled classes; intracellular signaling molecules, including MAP kinases, serine-threonine kinases, GTPase-activating proteins, and SH3/SH2 docking proteins. Marker genes from the cell cycle, Jak-Stat, NF{kappa}B, and P13/AKT pathways are included as well. Total RNA from experimental samples was used to synthesize 33P-labeled cDNAs by reverse transcription, according to protocols described in http://www.grc.nia.nih.gov/branches/rrb/dna.htm. All three array types were simultaneously hybridized with {alpha}-33P-dCTP-labeled cDNA probes overnight at 55°C in 4 ml of hybridization solution. Hybridized arrays were rinsed in 50 ml of 2xSSC and 1% SDS twice at 55° for 15 min each. The microarrays were exposed to phosphorimager screens for 1–3 days. The screens were then scanned in a Molecular Dynamics STORM PhosphorImager (Sunnyvale, CA) at 50 µm resolution. ImageQuant software (Molecular Dynamics, Sunnyvale, CA) was used to convert the hybridization signals on the image into raw intensity values, and the data thus generated were transferred into Microsoft Excel spreadsheets predesigned to associate the ImageQuant data format to the correct gene identities.

Data processing
Raw intensity data for each experiment was normalized independently for each array type by Z score transformation as described previously (27) . Briefly, intensity data are first, log10 transformed and then used for the calculation of Z scores. Z scores are calculated by subtracting the average gene intensity (within a single experiment) from the raw intensity data for each gene, and dividing that result by the standard deviation of all the measured intensities. Significant changes in gene expression were calculated by the Z ratio method (28) . In brief, gene expression differences between any two experiments are calculated by taking the difference between the observed gene Z scores. Z ratios then express these differences in terms of their relationship to the standard deviation of the distribution of all of the observed gene changes. The calculated Z ratios for a given comparison are rank ordered on this basis and a Z ratio of ±1.96 is inferred as significant (p<0.05).

DSCR1(Adapt78) antibody and Western blot analysis
Using the predicted open reading frame sequence of DSCR1(Adapt78), we designed a C-terminal 16-amino-acid peptide that was used as an antigen to immunize rabbits. We added a cysteine to the N terminus of this peptide (KIIQTRRPEYTPIHLS) and then conjugated the peptide to keyhole limpet hemocyanin. Polyclonal antibodies were raised commercially (ProSci, Inc., Poway, CA) by injection of this complex into rabbits. Serum from one of the immunized rabbits was affinity purified on a column with the covalently attached DSCR1(Adapt78) peptide that was used for immunization. Western blot analysis was performed, following the protocol from ProSci, Inc., using an ECL-detection system from Amersham. Final dilution of the purified antibody for Western blot analysis was 1:500. Our antibody specifically recognized calcipressin1 (the dscr1/adapt78 protein) as a single band.

Sod 1 assay
Activity of the sod 1 enzyme (intracellular Cu,Zn SOD) was measured by the cytochrome c reduction assay as described in (29) . Cells were disrupted in water and whole homogenates were used for assays. Total concentration of protein in homogenates was measured using the protein reagent from Bio-Rad Laboratories (Hercules, CA). The sod 2 enzyme was inactivated using dodecyl sulfate, which does not affect sod 1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Regulated expression of DSCR1(Adapt78) gene
We have previously shown that DSCR1(Adapt78) is highly expressed in brain, and predominantly in neurons (24) . Therefore, in the present studies, we used the neuron-like PC-12 cell line as a model to study the effects of regulated DSCR1(Adapt78) overexpression. The DSCR1(Adapt78) gene is normally expressed at low levels and only up-regulated when cells are exposed to stresses, such as oxidative stress or calcium overload (1 , 20 , 23 , 30 , 31) . It is maximally up-regulated ~5 h after stress is initiated and then gradually down-regulated to basal levels. Here, we also time-regulated DSCR1(Adapt78) expression to mimic its expression during stress conditions. The tet-off gene expression system (Clontech Laboratories, Inc.), which employed doxycycline regulated transgene expression, was used in these experiments (Fig. 1 A). We overexpressed a DSCR1(Adapt78) cDNA fragment corresponding to isoform 1 of the gene, which is predominantly expressed in neurons (23) . Cells were continuously grown in the presence of doxycycline to keep the transgene "silent," and inducible DSCR1(Adapt78) transgene expression was achieved by doxycycline withdrawal from the media. For repression experiments DSCR1(Adapt78) was first overexpressed by culturing cells for ~1 week in media without doxycycline, and then doxycycline was added (to inhibit the transgene) for 0, 1, 3, 6, 9, 24, or 48 h. Expression of the DSCR1(Adapt78) transgene was significantly down-regulated in 3 h and almost completely inhibited as early as ~6 h (Fig. 1B ).



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  		Figure 1. Regulated expression of the DSCR1(Adapt78) gene. A) Schematic of DSCR1(Adapt78) regulation using the tet-off system. In the absence of doxycycline tTA binds TRE and thereby activates transcription of the DSCR1(Adapt78) gene. B) Regulated expression of DSCR1(Adapt78) mRNA. Before the experiment, cells were continuously grown in the presence of doxycycline to keep the transgene "silent." Then inducible DSCR1(Adapt78) transgene expression was achieved by doxycycline withdrawal from the cell medium. After doxycycline had been withdrawn from the medium, we could achieve about a 10-fold increase in DSCR1(Adapt78) expression (not shown), as previously reported (23) . We next again repressed DSCR1(Adapt78) expression by doxycycline addition. For repression experiments doxycycline was added (to inhibit the transgene) for 0, 1, 3, 6, 9, 24, or 48 h. The level of DSCR1(Adapt78) RNA was significantly reduced by as early as 3 h, and in 6 h DSCR1(Adapt78) RNA almost completely disappeared. DSCR1(Adapt78) expression was analyzed using Northern hybridization. Membranes were first hybridized to the DSCR1(Adapt78) probe and then to a glyceraldehyde-3-dehydrogenase (GAPDH) probe to control for RNA loading levels. We have previously reported the stability of GAPDH RNA under the same conditions of DSCR1(Adapt78) regulated transgene expression (23) . Loading also was controlled by the amount of 28S and 18S RNA. Gels were stained with ethidium bromide, photographed under UV light, and the amount of 28S and 18S was evaluated.

Genes affected by DSCR1(Adapt78) down-regulation
Following the shutdown of DSCR1(Adapt78) transgene overexpression, we analyzed the time course of expression of other genes, using cDNA microarrays. Total RNA samples, isolated at different time points of DSCR1(Adapt78) transgene shutdown (Fig. 1 ), were used as microarray probes for Fig. 2 . Three different microarray types were used in this study: immunoarray, neuroarray, and signal transduction array (see Experimental Procedures). Each array was run in triplicate, and all experiments were repeated twice. Data were processed, normalized, and Z scores were calculated to evaluate expression of each gene as described in Experimental Procedures. Please, see SOD1 gene expression Z scores for example (Fig. 3 A).



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  		Figure 2. Genes significantly differentially expressed during DSCR1(Adapt78) down-regulation. Total RNA samples isolated at different time points of DSCR1(Adapt78) transgene shutdown as shown in Fig. 1 (0, 1, 3, 6, 9, 24, and 48 h) were used as microarray probes. Microarrays were analyzed as described in Methods. Results represent mean values from two experiments in which three determinations per point were made. Data were processed, normalized, and Z scores were calculated to evaluate the expression of each gene. Significant changes in gene expression were calculated by the Z ratio method as described in Methods. Black color represents low gene expression, red, high.



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  		Figure 3. SOD1 gene expression decreases following down-regulation of DSCR1(Adapt78). DSCR1(Adapt78) gene was down-regulated as shown in Fig. 1 . A) SOD1 mRNA. Relative levels of SOD1 mRNA were estimated directly from microarray data using the average Z scores ± standard errors from three independent measurements for each time point. B) Sod 1 protein. Protein expression was determined by the Western blot technique. The same membrane was probed with the sod 1 and calcipressin 1 antibodies; ß-tubulin was used as a loading control. Level of calcipressin 1 was significantly reduced as early as 6 h, and in 24 h, calcipressin 1 almost completely disappeared.

Although expression of most genes was unaffected, DSCR1(Adapt78) down-regulation caused up-regulated expression of some genes, down-regulation of others, as well as transient gene regulation (Fig. 2 ). It is interesting that among those transiently regulated was the gene encoding for DNA cytosine-5 methyltransferase. It was up-regulated at 1 and 3 h of DSCR1(Adapt78) down-regulation, its high levels of expression remained unchanged at 3, 6, and 9 h, and then it was decreased again at 24 h, and diminished to basal levels at 48 h. DNA cytosine-5 methyltransferase can methylate DNA and thus can down-regulate transcriptional activity of other genes. It is possible that regulation of DNA cytosine-5 methyltransferase is an early event through which DSCR1(Adapt78) may execute various transient responses to oxidative stress.

Somatostatin I precursor and several other mRNAs in PC-12 cells were gradually up-regulated from 0 to 48 h following DSCR1(Adapt78) down-regulation. In contrast, genes encoding superoxide dismutase 1 (SOD1), NF{kappa}B inducing kinase (NIK), and others, were gradually down-regulated. Note that the DSCR1(Adapt78) signal was also gradually decreased (Fig. 2 ), proving the reliability of our techniques. Remarkably, DSCR1(Adapt78) down-regulation caused down-regulated expression of several protein kinase-encoding genes. The DSCR1(Adapt78) product, calcipressin 1, was previously demonstrated to antagonize the action of the serine/threonine phosphatase, calcineurin (3 , 4) , and here, we see that it can additionally regulate the expression of other protein kinases. In the present study, we have focused on the SOD1 gene; however, regulation of protein kinases and other genes by DSCR1(Adapt78) will be the subject of future studies.

DSCR1(Adapt78) regulates SOD1
SOD1 mRNA levels were gradually down-regulated following shutdown of DSCR1(Adapt78) transgene overexpression (Fig. 3A ). This also caused lowered production of sod 1 protein (Fig. 3B ). On the basis of this observation, we hypothesized that DSCR1(Adapt78) might also induce SOD1 expression. We next overexpressed the DSCR1(Adapt78) transgene and found that production of sod 1 protein was indeed up-regulated when DSCR1(Adapt78) was overexpressed (Fig. 4 ). Using Western blot analysis, we estimate that, following 24 h of DSCR1(Adapt78) overexpression, sod 1 protein levels were ~78% higher than they were before DSCR1(Adapt78) overexpression (Fig. 4 ). These results were further confirmed by comparison of sod 1 enzyme activity in cells overexpressing calcipressin 1 with control cells (Fig. 4D ). Sod 1 activity was up-regulated ~70% following 24 h of DSCR1(Adapt78) overexpression. Considering that sod 1 levels in the brains of Down’s syndrome patients are elevated by ~50%, compared with controls (32) , this is very significant increase.



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  		Figure 4. DSCR1(Adapt78) stimulates expression of the SOD1 gene. First, cells were grown in the presence of doxycycline to keep the DSCR1(Adapt78) transgene "off." To overexpress DSCR1(Adapt78), complete medium then was replaced with medium lacking doxycycline for 0, 3, 6, and 24 h. A) Representative Western blot showing sod 1 protein stimulation by calcipressin 1. The same membrane was probed with the sod 1 and calcipressin 1 antibodies; ß-tubulin was used as a loading control. Calcipressin 1 was significantly overexpressed as early as 3 h and reached maximal expression in ~6 h. B) Summary of sod 1 protein stimulation by calcipressin 1. X-ray films were quantified using IPLab software (Scanalytics) and adjusted according to the loading. Sod 1 and calcipressin 1 protein levels are expressed in arbitrary units (A.U.). The figure represents results of three independent experiments. Sod-1 protein levels were elevated 78 ± 6% after 24 h of DSCR1(Adapt78) overexpression. C) Western blot showing calcipressin 1 and sod 1 production in parental PC-12 cells. This control experiment was performed as described in panel A. Cells were grown in the presence of doxycycline, and complete medium then was replaced with medium lacking doxycycline for 0, 3, 6, and 24 h. Membranes were stained with Ponceau reagent to confirm equal loading. D) Stimulation of sod 1 activity by calcipressin 1. Calcipressin 1 was overexpressed in PC-12 cells as shown in panels A and B. Total protein was extracted from the cells, and sod 1 activity was measured as described in Methods. Bars represent mean values of triplicate samples from two experiments ± standard errors. Sod 1 activity is expressed in units per one microgram of total protein. Activity of sod 1 before DSCR1(Adapt78) overexpression was 13 ± 2, and it was increased to 22 ± 3 following DSCR1(Adapt78) overexpression (78% higher). This difference was statistically significant at the P <= 0.05 level by the student’s t test (one population).

To demonstrate that induction/down-regulation of SOD1 is not due to the doxycycline withdrawal from/addition into the cell culture medium, we ran similar experiments using the parent PC-12 cell line, in which the plasmid carrying our DSCR1(Adapt78) transgene was missing. The same doxycycline concentration in the medium was used (0.02 µg/ml). Doxycycline did not affect either calcipressin 1 or sod 1 protein levels in these cells (Fig. 3C ), confirming that SOD1 was regulated by DSCR1(Adapt78), and not by doxycycline.

It seems that DSCR1(Adapt78) expression in cells is tightly regulated. This gene is normally expressed at a low level, and it is induced by stress. Its expression is rapidly turned off when stress is withdrawn. Similarly, DSCR1(Adapt78) is feedback-regulated in our expression system. In the PC-12 cells used in our study, its level rose after withdrawal of doxycycline from the cell growth medium, then it reached maximal levels in ~6 h, and later was slightly decreased but remained above the endogeneous level (Fig. 4 ). It is likely that cells recognize and down-regulate high levels of DSCR1(Adapt78) expression, which may be harmful for them. Sod 1 protein levels (Fig. 4 ), however, continued to gradually rise, even when calcipressin 1 levels were down-regulated. Rapid regulation of DSCR1(Adapt78) expression is probably facilitated by both DSCR1(Adapt78) mRNA and protein (calcipressin 1) rapid degradation in cells. We estimate that DSCR1(Adapt78) mRNA half-life is not longer than 2 h (Fig. 1 ) and calcipressin 1 half-life is not longer than 6 h (Fig. 3B ). The half-life of the sod 1 protein, in contrast, is much longer, around 24 h (33) depending on cell type. This might explain why sod 1 protein levels continued to rise at the 24 h time point, even when calcipressin 1 levels no longer increased.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings demonstrate that DSCR1(Adapt78) can stimulate production of the sod 1 enzyme. The levels of sod 1 protein were up-regulated as much as 78% by DSCR1(Adapt78), which is significant induction, considering that sod 1 levels in Down’s syndrome brains are elevated only ~50% compared with controls (32) . In turn, elevated levels of sod 1 may have two opposite effects. Sod 1 can normally reduce excessive levels of the superoxide radical and protect cells from oxidative stress. However, in cells without excessive levels of superoxide, sod 1 overexpression might cause more damage than benefits, and its deregulated expression has been strongly associated with Down’s syndrome. Many studies have demonstrated that Down’s syndrome involves a high-sod 1 level in a variety of cell types and organs (32 , 34 35 36 37 38) . At the same time, catalase and glutathione peroxidases, which degrade the hydrogen peroxide produced by the action of sod 1, are not changed in Down’s syndrome. Such misbalance may cause higher steady-state levels of hydrogen peroxide, resulting in cell damage that may contribute to the pathogenesis of Down’s syndrome (39 , 40) .

Earlier, we (24) and others (3) observed that the DSCR1(Adapt78) gene, which is localized in the Down’s syndrome candidate region, is overexpressed in brains from Down’s syndrome patients. Because DSCR1(Adapt78) causes overexpression of SOD1, it is likely that an elevated level of sod 1 in Down’s syndrome brains might be due, at least in part, to DSCR1(Adapt78) overexpression. This hypothesis is also in agreement with the observation that both SOD1 (41) and DSCR1(Adapt78) (23 , 24) are highly expressed in the same type of brain cells—neurons. This fact also suggests that neurons might be the most vulnerable cells in Down’s syndrome, in which elevated levels of sod 1, due to the gene triplication, might be additionally magnified due to elevated levels of calcipressin 1. In fact, neurological pathologies are the most common manifestations in Down’s syndrome, and it seems that oxidative stress and neuronal damage occur already in utero (39) .

We have recently demonstrated that DSCR1(Adapt78) is also overexpressed in Alzheimer’s disease (24) . Down’s syndrome subjects commonly develop Alzheimer’s disease in their later years of life (42) and these two diseases might have common pathways. It is possible that DSCR1(Adapt78) overexpression also causes elevated levels of sod 1 and consequently damage in Alzheimer’s disease. This hypothesis, however, remains to be tested.

The mechanism by which DSCR1(Adapt78) may regulate SOD1 expression is unknown; however, there are indications that the DSCR1(Adapt78) protein, calcipressin 1, may also be a transcriptional factor. So far, calcipressin 1 has been demonstrated to bind to, and down-regulate the activity of calcineurin (3 , 4) . However, binding and regulation of calcineurin might be not the only function or even the major function of the DSCR1(Adapt78) gene product. A stretch of ~80 amino acids near the N terminus of DSCR1(Adapt78) protein family members shows similarity with an RNA recognition motif, which is found in many RNA binding proteins and in a few single-stranded DNA binding proteins, (43) suggesting that DSCR1(Adapt78) might be a transcriptional factor. Additionally, the dynamic of calcipressin 1 localization within the cells also indicates that it might be a transcriptional factor: When produced, it is transported from the cytoplasm, and it accumulates in the nuclear compartment (4) .

In conclusion, we have demonstrated a new pathway for the regulation of SOD1 expression via DSCR1(Adapt78). DSCR1(Adapt78) is known to be up-regulated during oxidative stress, and in disease states involving chronic inflammation. Our data may help to better understand mechanisms of sod 1 expression and its relationship to human diseases such as Down’s syndrome and Alzheimer disease.


   ACKNOWLEDGMENTS
 
We thank Dr. Daniel Rettori (School of Pharmacy, University of Southern California) for the measurement of sod 1 activity. This work was supported by grant # AG16256 from the NIH/NIA to K.J.A.D.


   FOOTNOTES
 
doi: 10.1096/fj.03-0451com

Received for publication June 24, 2003. Accepted for publication September 9, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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