A Sod2 Null Mutation Confers Severely Reduced Adult Life Span in Drosophila

Corresponding author: Atanu Duttaroy, Howard University, 415 College St., NW, Washington, DC 20059., aduttaroy{at}howard.edu (E-mail)

Communicating editor: J. BIRCHLER

	ABSTRACT

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A null mutation for the Sod2 gene, Sod2ⁿ²⁸³, was obtained in Drosophila melanogaster. Homozygous Sod2 null (Sodⁿ²⁸³/Sodⁿ²⁸³) adult flies survive up to 24 hr following eclosion, a phenotype reminiscent of mice, where Sod2^-/- progeny suffer neonatal lethality. Sod^n283/+ heterozygotes are sensitive to oxidative stress induced by paraquat treatment.

THE enzyme manganese superoxide dismutase, also known as Sod2, detoxifies superoxide radicals (O^-₂) in mitochondria. O^-₂ is a byproduct of oxidative phosphorylation in all aerobic organisms. In the case of higher eukaryotes, with relatively more oxygen demand, loss of Sod2 activity causes pleiotropic phenotypes affecting the heart, brain, muscle, and behavior, culminating in neonatal lethality (reviewed by WALLACE 2001 ). Fundamentally, loss of Sod2 function causes a net increase in O^-₂ load in mitochondria, resulting in mitochondrial membrane damage, which eventually leads to cell death in key tissues like the muscle, heart, and liver (MELOV et al. 1999 ; KOKOSZKA et al. 2000 ). Mitochondrial pathologies in Sod2 null mice are suggestive of human conditions like Friedrich's ataxia and 3'-hydroxy-3-methyl CoA lyase deficiency (MELOV et al. 1999 ). The Drosophila Sod2 gene has been isolated and characterized (DUTTAROY et al. 1994 , DUTTAROY et al. 1997 ), but no mutant for Sod2 function has been obtained because of the apparent absence of a deletion straddling the Sod2 locus (FLYBASE 1999 ). As a first step toward understanding the molecular etiologies associated with the loss of Sod2 function in specific tissue or cell types, we isolated a null mutant for the Sod2 gene in Drosophila.

A P insertion called KG06854 was identified in the 5'-untranslated region that is located 102 nucleotides upstream from the Sod2 translation start site in Drosophila (Drosophila P-insertion screen/gene disruption project). Adults homozygous for KG06854 are completely viable and fertile. To obtain a null mutant for the Sod2 gene, we used standard techniques whereby KG06854 was excised using P(2-3), which is the transposase producer (ROBERTSON et al. 1988 ). Putative excision events were selected on the basis of their loss of the white⁺ phenotype originally associated with KG06854 (ROSEMAN et al. 1995 ). Approximately 300 white-eyed males were selected, and independent lines were established for the purpose of analyzing the Sod2 gene (Fig 1). Each putative excision line is expected to carry both straight wing (homozygous) and curly wing (heterozygous) flies at a ratio of 1:2; however, in line 283, this ratio was 1:9 (Fig 1). We therefore measured the viability of 283 homozygotes collected within 3 hr of eclosion and monitored their viability every 12 hr. A large majority of these adults (98%) died within 24 hr, and by 36 hr all homozygotes had perished (Fig 2). Since Sod2 knockout mice exhibit neonatal lethality (LI et al. 1995 ; LEBOVITZ et al. 1996 ) and Sod2 knockdown in Drosophila reduces its life span (KIRBY et al. 2002 ), we investigated the condition of the Sod2 gene and its expression in line 283. For this purpose an in-gel assay was used that is capable of monitoring the activity of both Sod1 and Sod2 proteins, which appeared as two distinct bands (Fig 3). A similar assay with total protein extracted from 283 homozygotes showed no Sod2 enzyme activity, although their Sod1 activity remained normal (Fig 3A). Furthermore, 283 heterozygotes (283/Cy), homozygote KG06854, and Canton-S all exhibited perfectly normal Sod1 and Sod2 activity (Fig 3A). Even though 283 homozygous flies showed no Sod2 activity, either an inactive or a truncated Sod2 protein could still have been present. Western analysis with anti-Sod2 antibody (Stressgen Biotech, Victoria, BC) failed to show Sod2 protein in any form in the 283 homozygous flies (Fig 3B). This observation indicates that a bona fide null mutation for the Sod2 gene, called Sod2ⁿ²⁸³, has been obtained in Drosophila.

View larger version (21K): In this window In a new window Download PPT slide   Figure 1. Schematic of the genetic cross, used for excision of the KG06854 insertion. Putative excision events were analyzed following the establishment of stocks. In line 283, homozygotes for excision chromosomes were recovered far less frequently than their heterozygote sibs.

View larger version (19K): In this window In a new window Download PPT slide   Figure 2. Percentage survivorship among Sod2n283/Sod2n283, Sod2n283/+, and wild-type Canton-S at 25° on normal yeast cornmeal media. A total of 50% of Sod2n283/Sod2n283 flies died within 9 hr, and by 12 hr only 32% survived, compared to the Sod2n283/+ or Canton-S, very few of which died during that time.

View larger version (90K): In this window In a new window Download PPT slide   Figure 3. (A) In-gel enzyme assay for MnSOD activity on a 9% nondenaturing discontinuous PAGE gel. Nitro-blue tetrazolium (NBT, 1.23 mM) was used as substrate competing with SOD; 28 mM N',N',N',N'-tetramethylenediamine and 2.8 mM riboflavin were used as nascent free radical donors in a photochemical reaction that converts NBT to blue formazan. A prominent MnSOD band was observed in Canton-S, KG06854 homozygote, and Sod2n283/+ but was absent in Sod2n283/Sod2n283. Cu-Zn SOD was present at similar levels in all the strains. (B) Western blot assay of MnSOD. Total protein extracts were run in a 9% SDS PAGE minigel system and probed with 0.2 µg/ml rabbit anti-MnSOD polyclonal primary antibody (1:5000) and with goat anti-rabbit IgG HRP conjugate secondary antibody (1:50,000; Stressgen Biotech). Signal detection was done using electrochemiluminescence (Amersham Biosciences, Arlington Heights, IL). A 24-kD band specific for MnSOD was observed in wild-type Canton-S, KG06854 homozygote, and Sod2n283/+, but was absent in Sod2n283/Sod2n283.

Could the reduced life-span phenotype of the Drosophila Sod2ⁿ²⁸³ allele have resulted from inactivation of Sod2, or may some other gene(s) be affected? Because Sod2ⁿ²⁸³ was recovered through excision, other genes located in the vicinity of KG06854 could be affected. Southern analysis indicated that no genomic sequence other than Sod2 was affected in Sod2ⁿ²⁸³ (Fig 4). KpnI-digested genomic DNA prepared from Sod2ⁿ²⁸³/Cy heterozygotes, KG06854 homozygotes, and Canton-S flies showed a 2.0-kb band in the wild-type and Sod2ⁿ²⁸³/Cy lanes when Sod2 DNA was used as a probe (Fig 4). Interestingly, Sod2ⁿ²⁸³/Cy heterozygotes picked up an additional band that is 0.53 kb smaller than the 2.0-kb KpnI band. Our analysis revealed that this 1.47-kb band is formed because a large segment of DNA from the P-element 3'-end and a portion of the Sod2 gene are deleted in Sod2ⁿ²⁸³, resulting in the formation of this fusion fragment. Thus, the deletion did not extend beyond the Sod2 sequence, because the new 1.47-kb band is still recognizable by a Sod2 probe, so no other gene downstream to Sod2 is affected in Sod2ⁿ²⁸³. Furthermore, on the 5'-end of the P element, a diagnostic PCR band was amplified from Sod2ⁿ²⁸³, KG06854, and Canton-S DNA when all are in homozygous condition (Fig 4). This observation nullifies the possibility that the P-element 5'-end is affected in Sod2ⁿ²⁸³ during excision. Finally, we confirmed that the reduced longevity phenotype in Sod2ⁿ²⁸³ is exclusively due to the loss of Sod2 function by rescuing this phenotype with an MnSOD transgene that is located on the third chromosome (MOCKETT et al. 1999 ). More than 97% of Sod2ⁿ²⁸³ vg/Sod2ⁿ²⁸³ vg flies with a recognizable vestigial wing phenotype and carrying the MnSOD transgene on the third chromosome (red eyed) have been found to survive at least 96 hr posteclosion (Table 1). On the other hand, their Sod2ⁿ²⁸³ vg/Sod2ⁿ²⁸³ vg sibs without the MnSOD transgene (white eyed) die within 24 hr (Table 1). This led us to conclude that the reduced life-span phenotype of Sod2ⁿ²⁸³ resulted from the loss of the Sod2 sequence.

View larger version (36K): In this window In a new window Download PPT slide   Figure 4. Southern analysis with Sod2 probe shows a 2.0-kb KpnI band that appears in Canton-S. In Sod2n283/Cy heterozygotes, a second band of 1.47 kb appears due to the loss of the majority of the KG06854 element. Since this 1.47-kb excision product is still probable with the Sod2 probe, it proves that part of the Sod2 gene is actually affected in Sod2n283. Inset shows that the genomic DNA adjacent to the 5'-end of the P element remains intact in Sod2n283, since primer A (5'-TGTTTCCAGGAGAGGTTGCT-3') and B (5'-TCGAAAGACCCCAATCAGTC-3') amplify a same-size PCR product in homozygous Sod2n283 (lane 1), KG06458 (lane 2), and Canton-S (lane 3) flies.

A Drosophila mutant for Cu-Zn superoxide enzyme, also known as Sod1, shows hypersensitivity to oxidative stress condition when Sod1 homozygotes are fed paraquat (methyl violgen; PHILLIPS et al. 1989 ). Since homozygous Sod2 flies die so quickly, we tested the susceptibility of Sod2ⁿ²⁸³ heterozygotes under oxidative stress conditions by feeding them paraquat. When fed 10 mM paraquat, 50% of Sod2ⁿ²⁸³ heterozygotes survived after 24 hr of treatment, whereas 50% of wild-type flies survived for 60 hr (Fig 5). This indicates that Sod2ⁿ²⁸³ heterozygotes are approximately twice as sensitive to oxidative stress as wild-type flies.

View larger version (19K): In this window In a new window Download PPT slide   Figure 5. Survival curve for Sod2n283 heterozygotes and wild-type Canton-S at 25° on 10 mM paraquat and sucrose media. In Sod2n283 heterozygotes, 50% survival was observed after 24 hr compared to 60 hr in Canton-S flies. To compare this effect in a comparative genetic background, we also tested KG06854/Cy flies as a second control. These flies are significantly less sensitive to paraquat treatment compared to Sod2n283/Cy flies, establishing the point that reduced Sod2 activity actually made the Sodn283 heterozygotes more sensitive to oxidative stress.

In summary, we showed that a complete loss of Sod2 function in Drosophila leads to an extreme reduction in life span. This corroborates findings from Sod2 null mice, which also suffer early lethality. By contrast, multiple pathophysiological conditions were reported due to the loss of Sod1 function in Drosophila, including reduced life span (PHILLIPS et al. 1989 ), signs of premature aging (ROGINA and HELFAND 2000 ), and degeneration (PHILLIPS et al. 1995 ), whereas Sod1 knockout mice are relatively healthy and show no sign of degeneration (REAUME et al. 1996 ). It is possible that the lack of a third SOD enzyme, the extracellular SOD, makes the insect cells more reliant on SOD1 activity. The enzyme Sod2, active in mitochondria, carries the maximum load of detoxifying superoxide radicals, because 97% of superoxides are generated in mitochondria. Thus, the absence of Sod2 activity may affect the life span in all organisms, because Sod2 function is more crucial to aerobic organisms. Extension of the average life span by overexpression of Sod2 in postmitotic cells (SUN et al. 2002 ) and in motor neurons (J. P. PHILLIPS, T. L. PARKES and A. J. HILLIKER, personal communication) further supported this notion. However, these observations are not totally unequivocal, since transgenic overexpression of MnSOD beginning early in development has no beneficial effect on longevity in Drosophila (MOCKETT et al. 1999 ).

	ACKNOWLEDGMENTS

The authors are indebted to William Orr for providing the MnSOD transgenic line. The P-element insertion line KG06854 was obtained from the Drosophila Stock Center in Bloomington, Indiana. Work was supported by a grant (1R15AG17846-01) from the National Institutes of Health to A.D.

Manuscript received April 14, 2003; Accepted for publication August 26, 2003.

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