Anoxia regulates gene expression in the central nervous system of Drosophila melanogaster

Molecular Brain Research 46 Ž1997. 325–328

Short communication

Anoxia regulates gene expression in the central nervous system of Drosophila melanogaster Enbo Ma, Gabriel G. Haddad

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Departments of Pediatrics (Section of Respiratory Medicine) and of Cellular and Molecular Physiology, Yale UniÕersity School of Medicine, PO Box 208064, 333 Cedar Street, New HaÕen, CT 06520–8064, USA Accepted 22 January 1997

Abstract We took advantage of the Drosophila melanogaster’s extraordinary resistance to anoxia to study the molecular mechanisms underlying this phenomenon. We analyzed mRNA expression of heat shock proteins ŽHSP. ŽHSP26 and HSP70., ubiquitins ŽUB. ŽUB3 and UB4., cytochrome oxidase I ŽCOXI. and superoxide dismutase ŽSOD. using slot blot analysis. The expression of HSP genes, especially HSP70, was remarkably up-regulated Žup to a thousand-fold. while those of UB4 and COXI were down-regulated Ž10–60%. in response to the anoxic stress. The expression of UB3 gene was up-regulated by 1.5 = and the expression of SOD gene was not significantly affected. In response to heat shock stress, the expression of HSP genes increased by up to several thousand-fold, the expression of UB genes increased modestly Ž23–91%. but the expression of SOD and COXI genes was reduced by 25%. Furthermore, the expression patterns of HSP genes under anoxia and heat shock were clearly different. The expression of HSP genes peaked by 15 min into anoxia and then declined but stayed above baseline. In contrast, their expression increased as a function of time during heat exposure. From these results, we conclude that: Ž1. different forms of stress regulates gene expression in different ways; Ž2. anoxia differentially regulates gene expression; and Ž3. the up-regulation of HSP70 and down-regulation of UB4 by anoxia are consistent with the idea that Drosophila melanogaster resist anoxia, at least in part, by protecting proteins against degradation. ‘‘ 1997 Elsevier Science B.V. All rights reserved. Keywords: Heat shock protein ŽHSP.; Ubiquitin ŽUB.; Superoxide dismutase ŽSOD.; Cytochrome oxidase ŽCOX.; Anoxia; Drosophila; Slot blot; mRNA

There is a growing body of evidence that shows that O 2 deprivation damages cells through necrosis or apoptosis and via processes that involve protein degradation and lipid peroxidation. Such processes generally occur in mammals and anoxia-sensitive organisms w1,20,21,25,33x. We have recently shown that, unlike mammalian tissues in general and mammalian nervous systems in particular, fruitflies Drosophila melanogaster are extraordinarily resistant to anoxia and can survive hours and recover after a total lack of O 2 . How these flies survive anoxia, protect themselves and succeed in preventing tissue injury is not clear. The molecular mechanisms underlying the basis for this tolerance are not studied in this animal. Since Ž1. there is a major difference in anoxia tolerance between fruitflies and mammals w13,18,32x and Ž2. O 2 deprivation is well-known to regulate gene expression, such as erythropoeitin w11,28x and tyrosine hydroxylase

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Corresponding author. Fax: q1 Ž203. 785-6337.

w6,24x, we hypothesized, as a first step, that Drosophila induces the synthesis of essential proteins to survive anoxia. We chose to study the regulation of four such genes aiming at specific important pathways for cell survival under stress in the central nervous system ŽCNS. of the fruitflies. The proteins encoded by these genes are related to either protein protectionrdegradation, oxygen radicals scavenging or enhancement of respiratory chain enzymes and oxidative capacity. Drosophila stock. Wild-type D. melanogaster flies ŽCanton-S. were maintained at room temperature Ž218C. on standard food media consisting of 82.5% water, 6.5% cornmeal, 0.74% agar, 1.6% yeast and 8.7% molasses. Either 0.56% propionic acid or 0.87% Tegosept was added to prevent the growth of mold. Anoxia and heat shock. Flies were collected into three 50-ml Corning tubes: one was used as control Žnormoxia: room air., one was subjected to anoxia Ž- 0.02% of O 2 . and the rest was subjected to heat shock Ž378C. as a positive control and as stressful condition that could be

0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 0 7 4 - 0

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E. Ma, G.G. Haddadr Molecular Brain Research 46 (1997) 325–328

helpful in assessment of the specificity of the anoxic stress. The tubes containing the flies used for the anoxia experiments were covered with a nylon mesh and placed in a 1 liter plastic box connected to N2 . Following the introduction of N2 , the level of oxygen within the box Žmeasured with a Beckman oxygen analyzer. declined to - 0.02% within 1 min. For the heat shock experiment, flies were incubated in a 378C incubator for 15 min and 1 h, respectively. To prevent possible degradation of RNA, the flies from the three groups mentioned above were immediately frozen in liquid N2 after treatment. For large collections of fly heads, flies from those groups were vigorously shaken after being frozen in liquid N2 . Their heads were collected by passing them through a number of sieves of successively smaller pore size. Specifically, shaken flies were passed through two sieves ŽMINI-SIEVE INSERT-ASTD from Bel-Art Products, Pequannock, NJ; openings of 707 and 500 m m.. Most heads passed through 707 m m sieve and accumulated on the 500 m m sieve; most bodies and some intact flies were collected on the 710 m m sieve; antennae, very small embryos, halters and fragments of legs passed through 500 m m sieve. In order to obtain heads only, the collected heads after first sieve were once more subjected to the same sieves again. RNA isolation. Total RNA was extracted and purified from fly heads exposed to heat shock, anoxia and normoxia by the acid guanidiumrphenolrchloroform method w5x using TRIzol Reagent kit from Gibco-BRL ŽGaithersburg, MD.. PolyŽA.q RNA was purified from the above groups of fly heads by affinity chromatography on oligoŽdT.-cellulose w27x using FastTrack mRNA isolation kit from Invitrogen ŽSan Diego, CA.. Slot blot hybridization. To study the pattern of expression of the gene transcripts of interest, slot blot analysis was applied using specific oligonucleotide probes ŽODN. designed according to published sequences ŽCOXI 1132 – 1161 w8x, SOD 924 – 953 w29x, UB3524 – 553 and UB4 1715 – 1747 w19x,

Fig. 1. Slot blot hybridization. Six unique oligonucleotide probes were used to hybridize the blotted mRNAs from flies subjected to normoxia, anoxia Ž15, 60 and 240 min. and heat shock Ž15 and 60 min.. HSP, heat shock proteins; UB, ubiquitins; COXI, cytochrome oxidase; SOD, superoxide dismutase. Note: Ž1. the bands of normoxia for HSP mRNAs are very weak because these genes are barely active at 208C; Ž2. the expression for HSP26 in anoxic groups Ž15, 60 and 240 min. is also very low.

Table 1 Relative changes in mRNA levels during anoxia

HSP70 HSP26 UB4 UB3 COXI SOD

Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2

15 min

60 min

240 min

808.99 1743.01 2.84 56.59 1.01 0.91 1.55 1.47 0.72 0.74 0.96 1.01

494.82 1165.82 0.01 26.58 0.74 0.71 1.51 1.32 0.38 0.43 0.81 0.94

356.55 980.41 3.74 54.48 0.89 0.91 1.37 1.14 0.65 0.59 0.89 0.98

mRNA levels during anoxia normalized to levels during normoxia. HSP, heat shock proteins; UB, ubiquitins; COXI, cytochrome oxidase; SOD, superoxide dismutase. Expt. 1, experiment 1; Expt. 2, experiment 2. Note: the large difference between experiments 1 and 2 shown in this table for HSPs is mostly due to the extremely low expression during normoxia.

HSP26 16 – 45 w30x, HSP70601 – 630 w16x.. Briefly, certain amount of polyŽA.q RNA from each group were denatured by formaldehyde before blotting onto Nytran membranes ŽSchleicher and Schuell.. 3 h after pre-hybridization in 40% formamide and 4 = SSC, blots were hybridized with 32 P tail-labeled ODN probes at 448C overnight. After a final wash at 558C for 30 min in 0.2 = SSC in the presence of 0.1% SDS, membranes were exposed to X-ray film ŽKodak X-OMAT, Eastman Kodak. and subjected to autoradiography. Image analysis. The resulting autoradiograms were analyzed by means of a computerized imaging system ŽImageQuaNT, Molecular Dynamics.. The units used in this analysis are arbitrary. HSP. To study the effects of anoxia on the expression of HSP genes and to determine their patterns of expression under different forms of stress, we treated the flies with either anoxia or heat shock. Interestingly, anoxia and heat shock regulated the expression of HSP genes in different ways. Data in Fig. 1A and Table 1 showed that HSP, especially HSP70, were significantly up-regulated during severe anoxia up to a thousand-fold. HSP70 mRNA reached a maximum level within the first 15 min of anoxia and then decreased after 1 h to a steady level that was still highly above baseline ŽTable 1.. In contrast, both HSP70 and HSP26 mRNAs were remarkably increased as a function of time by heat shock treatment up to few thousandfold ŽFig. 1A, Table 2.. Although the increase of HSP70 mRNA caused by both anoxia and heat shock is likely to occur through activation of heat shock transcription factors w2,22x or possibly, though unlikely, triggered by the depletion of intracellular ATP w17x, the regulatory mechanisms involved in heat shock vs. anoxia may be different based on the findings in this study. Such differences could be due to the use of different sets of multiple cis-acting promoter elements or to different heat shock factors w23x. Regardless

E. Ma, G.G. Haddadr Molecular Brain Research 46 (1997) 325–328 Table 2 Relative changes in mRNA levels during heat shock

HSP70 HSP26 UB4 UB3 COXI SOD

Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2

15 min

60 min

6710.61 9186.91 136.97 1390.21 1.23 1.31 1.45 1.37 0.79 0.76 1.01 0.98

9304.84 12277.91 601.79 4508.78 1.91 1.44 1.24 0.95 0.74 0.74 0.79 0.78

mRNA levels during heat shock normalized to levels during normoxia. HSP, heat shock proteins; UB, ubiquitins; COXI, cytochrome oxidase; SOD, superoxide dismutase. Expt. 1, experiment 1; Expt. 2, experiment 2. Note: again the large difference between the two experiments is mostly due to the very low level during normoxia.

of the mechanisms of induction, however, we believe that HSP, especially HSP70, play a protective role against degradation of proteins caused by anoxia in the fly. This is based on Ž1. the fact that fruitflies show a differential regulation between HSP70 and HSP26 and Ž2. numerous studies have shown that HSP are chaperones that help protect cell and membrane function during stress w3x. UB. UB showed a mixed response to anoxia. UB4 was down-regulated during anoxia by 10–30% while UB3 was up-regulated by 1.4 = ŽFig. 1B, Table 1.. Heat shock up-regulated the expression of UB by 1.23–1.91 = ŽTable 2.. These results differed from the study of rat CNS which showed a relatively constant level of UB mRNAs after hypoxia w12x. We raise the question, therefore, as to whether this difference in UB expression is related to the difference in stress sensitivity of rat vs. fruitfly. The result of downregulation of UB4 during anoxia is interesting because UB4 and not UB3 plays a major role in accelerating protein degradation by unfolding target proteins w10x. Therefore, the down-regulation of UB4 in flies may indicate that the rate of degradation of certain proteins is likely to slow down during anoxia. This leads us to suggest that the down-regulation of proteins essential for degradative processes may be another mechanism that could be working in tandem with HSP70 to protect flies during anoxic stress. COXI and SOD. Because COX plays a key role in oxidative phosphorylation and SOD Žone of antioxidant enzymes. in scavenging toxic derivatives of O 2 w31x, we tested their responses to anoxia and heat shock. The results showed that the expression of COXI gene was down-regulated by up to 60% while that of SOD was not changed during anoxia ŽFig. 1C, Table 1.. Heat shock did not significantly down-regulate the expression of these two genes ŽTable 2.. The down-regulation of COXI during O 2 deprivation may indicate that there was less need for the

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transfer of electrons in the respiratory chain in the absence of O 2 . Although not conclusive, our observation that the expression level of SOD gene remained relatively stable during anoxia probably reflects the lack of an increase in superoxide radicals during anoxic stress. This is consistent with the fact that oxygen free radicals are mainly generated during reoxygenation and not during anoxia or hypoxia w4,15,34x. The constant level of SOD mRNA during hypoxia or ischemia was also found in other studies w9,26x although repeated ischemia may enhance the expression of Mn-SOD gene w7x. It is well-known that, during severe hypoxia in mammals and other lower animals, cellular protein synthesis decreases w14x. This is consistent with a design that would lower energy demands in the cell. Although this seems desirable from the point of view of global energy needs in cells, the synthesis of some proteins is not lowered. On the contrary, some proteins are presumably so essential for survival or cell recovery that they get up-regulated as we have seen in this work and other studies w24,28x. Hence, we believe that there is a set of synthetic ‘priorities’ that cells attemp to meet. We term this a ‘hierarchical strategy’ designed to protect cells against anoxia. This strategy seems to take place in Drosophila and it is likely that it plays an important role in the overall resistance to anoxia and possibly other stresses. In summary, anoxia differentially regulates gene expression. Different stresses regulate genes in different patterns. Anoxia up-regulated HSP many-folds while it down-regulates UB4 and COXI. UB3 was slightly up-regulated during anoxia while SOD seemed not to be affected by anoxia. Heat shock significantly regulates HSP ŽHSH70 and HSP26. while it has minor effect on others. The data from our study clearly support the idea that protective mechanismŽs. exists during anoxia in the anoxia-tolerant flies although these mechanisms do not rule out the existence of other potentially important mechanisms for anoxia tolerance.

Acknowledgements This study was supported by Donaghue Medical Research Foundation and HD 32573. We are grateful to Mr. Deren Shao for his technical assistance.

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