Communication Vol. 269, No. 36, Issue of September 9, pp. 22463-22465,1994 THE JOURNAL OF BIOLCCICAL CHEMISTRY

0 1944 by The American Society for Biochemistry and Moleeular Biology, Inc. Printed in U.S.A.

Mice with Duplications and Deletions at the Tme Locus Have Altered MnSOD Activity*

(Received for publication, June 8, 1994)

Endi Wang and Gin0 Cortopassi From the Institute for Toxicology, Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033

Superoxide radicals that result from normal cellular metabolism have been implicated as a cause of multiple age-related degenerative diseases (Halliwell, B., and Gutteridge, J. M. (1990) Methods Enzymol. 186, 1-85; Harman, D. (1988) Mol. Cell. Biochem. 84,155-161; h e s , B. N., Shigenaga, M. K., and Hagen, T. M. (1993) Proc. Natl. Acad. Sei. U. S. A. 90,7915-7922). Manganese super- oxide dismutase (MnSOD) is thought to be the sole en- zymic scavenger of superoxide in mammalian mitochon- dria. We have investigated MnSOD activity and gene dose in mice with deletions and a duplication of the Tme (t-associated maternal efiect) locus on chromosome 17. We find that MnSOD activity is significantly correlated with gene dose in these animals; animals with heterozy- gous deletions of Tme have 50% of normal activity, and animals with a heterozygous duplication of Tme have 150% of normal activity. These ratios of activity appear to be systemic, as they were observed in brain, heart, skeletal muscle and liver. The results support the model that basal MnSOD activity is regulated solely by cis el- ements, in that variation in MnSOD activity caused by altered gene dose on one chromosome is not compen- sated by gene activity on the other. Since gene knock- outs of MnSOD have not yet been generated, the tlUbs and ThP animals may become useful models for those study- ing the role of mitochondrial superoxide in pathophysi- ological processes. Amodel for the maternal-lethal effect of Tme deletions is proposed.

It has been proposed that endogenously generated superox- ide radical may contribute to cellular and organismal degen- eration (3-6). Approximately 90% of oxygen utilized by mam- malian cells is consumed in mitochondria, and approximately 3% of mitochondrial oxygen escapes complete reduction to H,O, most of which becomes superoxide radical (7-9). Mitochondria have a specific scavenger of superoxide, a manganese-contain- ing superoxide dismutase (10).

Elevated superoxide levels have been linked to human de- generative disorders by several routes. Deficiencies in SOD' activity lead to motor neuron death and familial amyotrophic lateral sclerosis (1, 2). The mitochondrial toxin l-methyl-4- phenyl-1,2,3,6-tetrahydropyridine induces superoxide forma- tion and dopaminergic cell death and Parkinsonism in humans

* This work was supported by Grant AG11967 from NIA, National Institutes of Health. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must there- fore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: SOD, superoxide dismutase; PCR, po- lymerase chain reaction.

and animals (11, 12); these same dopaminergic cells exhibit the highest frequency of mitochondrial somatic mutation anywhere in the human brain (13, 14,29). Ischemia-reperfusion injury in hear t and brain is coincident in time and space with superoxide production in mitochondria, and animals given superoxide dis- mutase are resistant to reperfusion injury (15). Thus the intra- cellular control of mitochondrial superoxide may have impor- tant ramifications for the understanding of degenerative disease. Another important mitochondrial antioxidant mole- cule is glutathione; an inhibitor of glutathione biosynthesis causes extensive mitochondrial damage in the brain (16).

We have investigated MnSOD gene dose and enzyme activity in mice with duplications or deletions of the Tme locus.

MATERIALS AND METHODS

strains (C57BU6, Tt"", ThP, and tlub2) were purchased from the Jackson Mouse Strains and Enzyme Activity Measurements-Mice of four

Laboratory. Tt"" mice bear a heterozygous duplication of the Tme locus, and ThP and tlub2 animals bear a heterozygous deletion of Tme (Fig. 1). Tissues were dissected and Dounce-homogenized in 50 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA, dissolved in six 5-s bursts with a Heat Systems sonicator, and centrifuged at 10,000 x g for 10 min; protein concentration was determined as in Refs. 17 and 18. SOD activity was measured in gels by the method of Beauchamp and Fridovich (19) except that samples were mixed 1:l with Histopaque (Sigma) to minimize band streaking (20). Activity in bands was meas- ured by laser densitometry. Soluble SOD activity was measured spec- trophotometrically as in Ref. 21. MnSOD activity was defined as total SOD activity minus activity in 5 mM NaCN.

Quantitatiue PCR-Gene dosage measurements in the T t o r ' , C57BL and tLUb2 strains were carried out by the method of Gilliland et al. (221, using genomic DNA purified from livers of each strain. The sequences of the primers 5' to 3' were as follows: 2.1, ccgctgctggggattgac; 2.2, gcccg- gagcctggccactc; and 2.3, gcccggagcctggccactcaatgcggcc. Primer 2.1 and 2.3 were used to synthesize a 177-base pair mutant product with the sequence gccggt converted to an EagI restriction site gccggc; this mu- tant product was used as a competitor of the wild-type template (22). Primers 2.1 and 2.2 were used to amplify the wild-type template, the exon 5 region of the MnSOD gene. PCR buffers were as described in Ref. 23. PCR conditions were initial denaturation at 94 "C using the "hot start" protocol (24) for 5 min, followed by 30 cycles of denaturation and annealing/extension at 94 "C for 30 s and 58 "C for 30 s, respectively, and last extension at 72 "C for 4 min. After PCR, products were digested by EugI and separated on 10% polyacrylamide gels. The PCR product of the wild-type template is EugI-indigestible; the mutant PCR product is EugI-digestible. Equivalence points (Fig. 2, arrowheads) are those at which the intensity of mutant product (for which a known amount of template was added) equals the intensity of the wild-type product (whose initial template concentration is unknown). Gels were photo- graphed and scanned using an HP ScanJet IIC scanner. Equivalence points were determined by using the NIH Image program on the digi- tized photographic images.

RESULTS AND DISCUSSION

Quantitative PCR Measurement of MnSOD Gene Dosage in Tme-variant Mice-The tlUb2 chromosome 17 contains a complex inversion and deletion of the Tme locus, which is near the MnSOD locus (Fig. 1); the Tt"" chromosome contains a complex inversion, and a duplication of the Tme region (25,26). Because the tlUb2 and Tt"" chromosomes are the result of a complex rearrangement, quantitative PCR measurements of MnSOD gene dose were undertaken. C57BL/6 mice are known to con- tain two copiesldiploid genome of the MnSOD gene. Relative to C57BU6, MnSOD gene copy number in Tt"" mice was esti- mated by quantitative PCR to be 2 x 413 = 2.7, near the value of 3 expected for a heterozygous duplication (Fig. 2, top and

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22464 MnSOD-variant Mice T qk Tme 11 MHC

_ I ' I I I ID

Wild type MnSOD I H

Tmeqk ? Y H C !f

t Chromosome

T

_ I 1 p b 2

;m, Tme c Tl

FIG. 1. Diagram of chromosomes 17 in "tor', ThP, and tLUbz ani- mals. Shaded DNA signifies the T chromosome. Gene symbols T, Qk, Tme, tf, and MHC signify the following loci: T, quaking, t-associated maternal effect, tufted, and multiple histocompatibility complex.

1 -1 C57BL

1

FIG. 2. Relative dose of MnSOD gene in three mouse strains. The mutant template produced by primer 2.1 and primer 2.3 was seri- ally diluted by 314 (left to rzgkrht) in the presence of 100 ng of genomic DNA. The top band of each panel is the PCR product from genomic template uncut by EagI; the lower band is the product from a known amount of mutant template cut by EagI. Arrows indicate the equiva- lence points at which input mutant product equals genomic product; equivalence points were confirmed by image analysis using NIH Image software.

center panels, lanes 9 and 10). Relative to C57BU6, quantita- tive PCR estimated MnSOD gene dose in t lub2 animals to be 2 x 9/16 = 1.1, very close to the value of 1 expected for a heterozy- gous deletion (Fig. 2, center and bottom panels, lanes 7 and 9). Thus, PCR results confirmed that MnSOD gene dosage is 3,2, and 1 in Tt'" heterozygotes, C57BW6, and t I u b 2 heterozygotes, respectively (Fig. 1).

Tme-variant Mice Have Deficiencies and Excesses of MnSOD but Not CuZnSOD Enzyme Activity-MnSOD and CuZnSOD isoezymes were separated electrophoretically and stained for activity (Fig. 3). Differences in MnSOD activity were clearly observed in Tme-variant animals. No change was observed in CuZnSOD activity. Gels were densitometrically scanned, and MnSOD peak integration showed that tLub2 and ThP animals have approximately 50% the MnSOD activity of C57BW6, whereas nor' animals have 150% (data not shown). The changes observed in MnSOD activity are consistent with the notion that basal MnSOD activity is solely regulated by cis- acting genetic elements, and that changes in total MnSOD activity mediated by mutations on one chromosome are not compensated by MnSOD gene activity on the wild-type sister chromosome.

Changes in MnSOD Level Are Systemic in Mutant Mice- Soluble MnSOD activity was assayed by the more precise spec- trophotometric assay in extracts of brain, soleus muscle, liver, and heart (21). MnSOD was about 50% of normal in heterozy- gous animals containing a deletion of the Tme locus, and about 150% of normal in animals with a duplication of the Tme locus (Table I). MnSOD activity was significantly correlated with gene dose in all tissues examined (Fig. 4). Correlation coeffi- cients for MnSOD gene dose versus activity for brain, soleus

MnSOD

CuznSOD

E. coli

Bovine

FIG. 3. SOD activity in skeletal muscle of three mouse strains. Samples from two soleus muscles were pooled, loaded in duplicate, and electrophoresed on 10% polyacrylamide gels. Each lane contains 100 pg of sample protein. Polaroid photographs of gels were scanned (Hewlett Packard ScanJet IIC). STD, pooled bovine CuZnSOD and Escherichia coli MnSOD standards (Sigma).

TABLE I MnSOD activity in control and Tme-variant animals

Values are the mean of three or more independent measurements. Unitdmg = units of MnSOD activity/milligram of protein. Normalized is the specific activity relative to C57BIJ6 mice. Skel M, soleus muscle.

Strain cz72 Tissue MnSOD activity

UniWme Drotein Normalized

C57BL 2 Heart 82.0 -.

1 Skel M 9.4 1 Liver 9.1 1 Brain 9.3 1

Tt(or1) 3 Heart 121.3 1.47 Skel M 14.8 1.57 Liver 13.2 1.46 Brain 11.4 1.23

T(hp) 1 Heart 26.3 0.32 Skel M 5.9 0.62 Liver 3.6 0.40 Brain 4.5 0.48

t(Lub2) 1 Heart 16.4 0.20 Skel M 3.2 0.34 Liver 2.9 0.32 Brain 5.4 0.58

01 I 0 1 2 3 4

Gene Dose

FIG. 4. Correlation between MnSOD activity and gene dose in multiple tissues. Each point represents the average of three or more measurements.

muscle, liver, and heart were 0.958, 0.951, 0.989, and 0.982. Toxicity to Egg Mitochondria May Explain the Unique Znher-

itance of Tme Alleles-tme is the only known mouse gene for which there is a maternal effect, i.e. deletions of the gene trans- mitted by sperm result in viable zygotes, but deletions trans- mitted through eggs result in fetal lethality (25, 26). Fetal mitochondria are thought to be derived almost completely from the egg (27, 28). Thus if mitochondria were oxidatively dam- aged in sperm bearing a deletion in MnSOD, such damaged mitochondria would not be contributed to the fetus. We propose that the Tme locus is MnSOD, and that deletion of MnSOD in haploid eggs may lead to oxidative damage which results in fetal lethality. The rescue of eggs with a deletion in Tme by sperm with a double dose of Tma (26) might also be explained by a reduction in zygotic oxidative damage.

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MnSOD-variant Mice 22465

Summary and Prospects-In conclusion, we have shown that mice with one, two, and three copies of the tme gene have systemic MnSOD activity in the ratio 1:2:3. These findings support the model that cis-acting regulators control basal Mn- SOD expression, and that trans-acting regulators do not com- pensate for activity differences in this range. A novel model to explain the maternal but not paternal lethality of deletions in the MnSOD gene is proposed. Such mutant mice may be of use to those studying the pathophysiological role of mitochondrial superoxide.

Acknowledgments-We thank E. Cadenas, C. Giulivi, J. Goin, and L. Washburn for helpful advice or mutant animals.

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E Wang and G Cortopassiactivity.

Mice with duplications and deletions at the Tme locus have altered MnSOD

1994, 269:22463-22465.J. Biol. Chem. 

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