1

Parkin mutant in the fly is largely rescued by metal-responsive 1

transcription factor (MTF-1) 2

3

Nidhi Saini1, Oleg Georgiev1, Walter Schaffner1*4

5

6

1Institute of Molecular Life Sciences 7

University of Zürich 8

Winterthurerstrasse 190 9

CH-8051, Switzerland 10

11

*Corresponding author: Walter Schaffner 12

Institute of Molecular Life Sciences 13

University of Zurich 14

Winterthurerstrasse 190 15

CH-8057 Zurich 16

Switzerland 17

phone: +41 44 635 31 50 18

fax: +41 44 635 68 11 19

Email: walter.schaffner@imls.uzh.ch 20

21

Running title: Parkin mutant largely rescued by MTF-1 22

word count for the introduction, results, and discussion sections: 3343 23

word count for the Materials and methods section: 2084 24

25

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.05207-11 MCB Accepts, published online ahead of print on 7 March 2011

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

2

Abstract 26

27

The gene for Parkin, an E3 ubiquitin ligase, is mutated in some familial forms of 28

Parkinson’s disease, a severe neurodegenerative disorder. A homozygous mutant of 29

the Drosophila ortholog of human parkin is viable but results in severe motoric 30

impairment including an inability to fly, female and male sterility, and a decreased 31

lifespan. Here we show that a double mutant of the genes for Parkin and the metal-32

responsive transcription factor MTF-1 is not viable. MTF-1, which is conserved from 33

insects to mammals, is a key regulator of heavy metal homeostasis and detoxification 34

and plays additional roles in other stress conditions, notably oxidative stress. In 35

contrast to the synthetic lethality of the double mutant, elevated expression of MTF-1 36

dramatically ameliorates the parkin mutant phenotype, as evidenced by prolonged 37

lifespan, motoric improvement including short flight episodes, and female fertility. At 38

the cellular level, muscle and mitochondrial structures are substantially improved. A 39

beneficial effect is also seen with a transgene encoding human MTF-1. We propose 40

that Parkin and MTF-1 provide complementary functions in metal homeostasis, 41

oxidative stress and other cellular stress responses. 42

43

Keywords: Drosophila/ MTF-1/ metal homeostasis/ parkin/ Parkinson’s disease 44

45

46

47

48

49

50

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

3

Introduction 51

52

Parkinson’s disease (hereafter referred to as PD) is the second most prevalent 53

progressive neurodegenerative disorder and the most common age-related movement 54

disorder (10, 13, 43, 59). Many molecular aspects of PD pathogenesis still need to be 55

clarified. Extensive studies point to oxidative stress as a major contributor to the 56

disease (28). Besides the gene for Parkin, an E3 ubiquitin ligase, four other genes: 57

PINK1, DJ1, UCHL1 and α-synuclein have been implicated in rare, early-onset, 58

familial forms of PD while LRRK2 is predominantly responsible for late onset PD (20, 59

57, 70). Much effort has gone into the development of animal models of PD, 60

including models in the fly Drosophila melanogaster. In our studies presented here, 61

we use a strain in which the ortholog of the human parkin gene has been disrupted by 62

insertion of a P-element transposon into the coding region (24, 48). 63

64

In mammals, the proteins PINK1 and Parkin cooperate to ensure proper quality 65

control of mitochondria and Parkin is particularly important for autophagy of faulty 66

mitochondria (reviewed in (8, 73). In agreement with this notion, Parkin deficient 67

flies suffer from mitochondrial malfunction (24, 45, 48), which distorts muscle 68

structure and causes severe locomotor defects and an inability to fly (24, 48). 69

Furthermore, both male and female parkin null mutant Drosophila are sterile (52), 70

exhibit an increased sensitivity to multiple stresses, including oxidative stress, and 71

have a reduced lifespan (23, 48). 72

73

Maintenance of metal homeostasis is an essential requirement for the proper 74

functioning of all organisms. An adequate supply of essential trace metals, like copper 75

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

4

and zinc, is important, whereas an excess can be highly toxic. Alterations in copper 76

homeostasis due to mutations in copper transporters cause Wilson’s and Menkes 77

disease (11, 31, 65). An imbalance in trace metal levels has also been implicated in 78

neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease, as well as 79

in senescence processes (42, 53, 54). To investigate the possible interplay of Parkin 80

function with metal homeostasis, we modulated the concentration of the metal 81

responsive transcription factor-1 (MTF-1) in parkin mutant Drosophila. MTF-1 is 82

conserved in evolution and its homologs have been characterized in humans (7, 36, 83

44), mice (25, 51, 69), fish (3, 9) and Drosophila (17, 60, 75). MTF-1, also referred to 84

as metal response element binding transcription factor 1, is a zinc-finger protein that 85

regulates transcription of its target genes by binding to DNA sequence motifs known 86

as metal response elements (MREs), which are typically located proximal to the 87

transcription start (12, 27, 32, 41, 64, 68). The majority of MTF-1 preferentially 88

localizes to the cytoplasm in quiescent, non-stressed cells but translocates to the 89

nucleus upon heavy metal load and a number of other stressful conditions (34, 58, 90

62). 91

92

Apart from counteracting the effects of heavy metal load, MTF-1 also induces 93

transcription of metallothionein genes in response to oxidative stress and infection (2, 94

21, 22). Metallothioneins (MTs), are small, cysteine-rich, metal-binding proteins with 95

a major role in metal homeostasis and detoxification (30, 46). MTs occur in all 96

eukaryotes, as well as in some prokaryotes. Heavy metals like zinc, copper and 97

cadmium are complexed by the cysteine sulfhydryl groups, which can also exert 98

antioxidant function (2, 29). In this context it is noteworthy that in a mouse PD model, 99

dopaminergic (DA) neurons of a MT-knockout mutant are more vulnerable to L-100

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

5

DOPA toxicity than neurons from mice with wild type MT (15, 39). This suggests 101

that MTs play a protective role against DA-quinone induced neurotoxicity. There are 102

more than ten functional metallothionein genes in humans, four in the mouse (50, 71) 103

and at least four in Drosophila, termed MtnA-MtnD (17, 40). The Drosophila MTs 104

are involved to different degrees in the defense against heavy metal stress. MtnA is 105

the most important under copper load, while MtnB preferentially binds cadmium and 106

protects against cadmium intoxication. MtnC and MtnD, despite sharing 67% amino 107

acid identity with MtnB, have only a minor role in protection against heavy metals, at 108

least when MtnA and MtnB are present (16, 18). The MtnA and MtnB genes (also 109

referred to as Mtn and Mto, respectively) are differentially regulated during 110

development (61). In addition to metallothioneins, MTF-1 also regulates, in 111

Drosophila, the expression of ferritins, the copper importer Ctr1B, the zinc exporter 112

ZnT35C, glutathione S-transferase and an ABC transporter (60, 63, 74). 113

114

MTF-1 proteins of human and Drosophila are highly similar in their DNA-binding 115

zinc finger region but quite divergent outside of it. Nevertheless, they can largely 116

complement each other in the protection against metal stress (6, 75). A major 117

difference between mammals and Drosophila is that metallothionein genes in 118

mammals are mainly induced by zinc and cadmium whereas in Drosophila they are 119

best induced by copper and cadmium (18, 75, 76). Moreover, disruption of the MTF-1120

gene in the mouse results in embryonic lethality (25), which is not the case for fly 121

mutants, which are viable and fertile. However, the fly mutants do display sensitivity 122

to cadmium, zinc and copper load as manifested by a reduced lifespan on heavy-metal 123

supplemented food, and also cannot tolerate copper starvation (17, 60). The 124

Drosophila allele MTF-1140-1R carrying a 4.1 kb deletion of the coding region has the 125

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

6

strongest phenotype and is considered a null mutation (17). We have used this allele 126

for experiments requiring a MTF-1 loss of function. 127

128

A link between cellular heavy metal handling and the parkin mutant phenotype was 129

suggested by our previous finding of a partial rescue of parkin mutant flies upon zinc 130

supplementation or chelation of redox-active metals (55, 56). In our present study we 131

set out to investigate the interaction between metal homeostasis and Parkin function, 132

or more specifically, MTF-1 and Parkin. We found that parkin mutants combined 133

with a knockout of MTF-1 are not viable, a genetic constellation termed synthetic 134

lethality. Parkin mutant Drosophila suffer from oxidative stress as a result of 135

heightened ROS production. A strong ubiquitous MTF-1 expression dramatically 136

ameliorates the parkin mutant phenotype: our results show that MTF-1 decreases 137

oxidative stress, normalizes concentration of essential trace metals, increases the 138

frequency of development to adulthood, restores female fertility, improves 139

muscle/mitochondrial morphology, locomotion and considerably extends the lifespan 140

of parkin mutant flies. 141

142

Materials and Methods 143

144

Fly food and maintenance 145

One liter of standard fly food was composed of 55 g cornmeal, 10 g wheat flour, 100 146

g yeast, 75 g glucose, 8 g agar and 15 ml anti-fungal agent Nipagin (15% in ethanol). 147

For the experiments of survival, development, eclosure frequency, ROS 148

measurements, real-time PCR and TEM muscle analysis, several conditions were 149

tested, namely, normal food (NF), NF supplemented with zinc chloride (4 mM) or N-150

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

7

acetylcysteine (15 mM) or bathocuproine disulfonate (BCS, copper chelator) (0.3 151

mM), and bathophenanthroline sulfonated sodium salt (BPS, iron (copper) chelator) 152

(0.1 mM). All flies were maintained at 25oC on a 12:12 hours light-dark cycle. 153

154

Construction of transgenic flies and fly stocks 155

UAS-MTF-1 flies were generated using the full-length MTF-1 cDNA cloned into the 156

pUAST vector. Tub-MTF-1 constructs were made by cloning the MTF-1 cDNA under 157

the control of the constitutive α–tubulin promoter. Both the constructs were injected 158

into the w1118 fly strain along with p( 2-3) helper plasmid and transformants were 159

selected based on the eye color (red/orange). The MTF-1140-1R null allele, generated by 160

homologous recombination, was characterized previously (17). The UAS-MTF-1, MT 161

(tub-MtnA), MTF-1140-1R strains were generated by recombination in our laboratory. 162

The w; +; tub-MtnA/TM6B (tub-MtnA), w; tub-MTF-1/Cyo;+ (tub-MTF-1) and 163

w;Actin-Gal4;UAS-MTF-1/TM6B (Actin-Gal4;UAS-MTF-1) flies were combined 164

with w;+;park25/25 (park25/25) by recombination. Unless specified otherwise, 165

w;+;park25/TM6B,w+ flies (heterozygous parkin mutants hereafter referred to as 166

park25/+) were used as controls for the w;+;park25/25 flies (homozygous parkin 167

mutants hereafter referred to as park25/25). park25/25 is a null mutation of the 168

Drosophila parkin gene (24). The TM6B balancer in the control flies was confirmed 169

to have no effect on any of the experiments performed (by removing it). 170

171

Lifespan determinations, eclosure frequency and fertility assays 172

For lifespan experiments, 1-2 day old flies (20 per vial) maintained at 25oC on a 12:12 173

hours light-dark cycle were examined for each genotype at least in triplicate. 174

Surviving flies were transferred to fresh food vials every 2 days and counted daily. In 175

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

8

the experiment with w;Actin-Gal4;UAS-MTF-1, park 25/TM6B flies, the survivors in 176

23 parallel independent sets were counted at regular time intervals. In each lifespan 177

assay testing different conditions, the controls of park25/+ and park25/25 flies raised on 178

NF were the same. The variations in the average lifespan of control flies in different 179

experiments can be attributed to subtle experimental variations. The metal chelator 180

concentrations selected for ROS determination had no significant effect on feeding 181

behavior of the flies (55). Fertility was assayed by placing single parkin mutant males 182

with 3-4 virgin yw females and by placing single virgin parkin mutant females with 183

two yw males. Vials were checked 3-7 days later for the presence of larvae and 184

eclosing adults. For the analysis of eclosure frequency, the number of days allowed 185

for egg laying and the parent population was the same in all vials of normal food (NF) 186

or zinc (Zn)-supplemented food and progeny flies were counted at the same time. 187

188

Behavioral assay (climbing performance and locomotion ability)189

The Climbing assay was performed as described (47). Flies of each genotype 190

(park25/+, park25/25, tub-MTF-1,park25/+, tub-MTF-1, park25/25) were anesthetized with 191

CO2 and individually counted and placed in food vials 24 hours before the assays 192

were performed to enable a full recovery from the effects of CO2. Ten flies were 193

placed in an empty 110-by 27-mm vial; a horizontal line was drawn 100 mm above 194

the bottom of the vial and another identical vial was used as a cover to provide more 195

mobile space. After the flies had acclimated for 10 min at room temperature, each 196

genotype was assayed in triplicate for five trials per set per genotype. The procedure 197

involved gently tapping the flies (on a soft surface) down to the bottom of the vial. 198

The flies were given 30 seconds to climb the vial and the number of flies which 199

crossed the 100 mm mark each time were recorded. These values were then averaged 200

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

9

and a group mean and standard error was calculated. The mean values of various fly 201

groups were statistically compared using an unpaired Student t-test. The study was 202

repeatedly performed with the same group of flies on every alternate day up to 10 203

days in an isolation room at 25oC, 60-70% humidity under standard lighting 204

conditions. Preliminary studies indicated no significant difference in the outcome of 205

climbing assays performed in normal light or red light conditions. 206

207

Fluorescent protein (EYFP) reporter208

The Drosophila metallothionein MtnA promoter (-446 to +74) was cloned from 209

genomic DNA using the primer pair 5’-CGG GAT CCA GGT ATG GGC TAT TTA 210

GGC C-3’ and 5’-GGG ATG GCC CCA AAG GAT CTG-3’ in a pCasper4-derived 211

vector carrying EYFP-coding sequence and the SV40 polyA site. Details were 212

reported previously (6, 18). Transgenics of MtnA-EYFP combined with parkin213

heterozygous or homozygous knockouts were made. Both fly types were then frozen 214

at the same age and photographs of EYFP expression were taken with a Leica MZ 215

FLIII fluorescence stereomicroscope and a Nikon Coolpix 950 digital camera (Leica, 216

Heidelberg, Germany) at an exposure of 730 ms. 217

218

RNA isolation and real-time analysis 219

Total RNA was purified from adult Drosophila tissue using the Nucleospin RNA II 220

protocol (Macherey-Nagel) and eluted in 60 μl of RNase-free water. cDNA was 221

prepared using the Transcriptor High Fidelity cDNA Synthesis kit from Roche. The 222

cDNA obtained was further purified using the AM 1906 Ambion DNA free kit and 223

used for analysis by real-time PCR on the Tecan Genesis 200/8 robot using the 224

Eurogenentec Mesa Green qPCR Mastermix Plus for SYBR assays. The qPCR run 225

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

10

was performed on an Applied Biosystem machine (ABI Prism SDS 7900 HT) in a 384 226

well format with a reaction volume of 10 μl. ΔΔct values were calculated by 227

subtracting the Δct calibrator from the Δct sample, Δct values were calculated by 228

subtracting Δct endogenous control from Δct target gene/calibrator. The normalization 229

strategy used has been described in Vandesompele et al (67). All the fold-change 230

values are normalized to respective park25/+ values on normal food (NF). The 231

housekeeping genes used were actin5c, TBP and GAPDH. Two sets of primer 232

sequences were used for each of the transcripts quantified: for parkin, the first primer 233

set was 5’-AAG ATC ATA TTT GCC GGT AAG GAA-3’ and 5’-CGC TTT GCT 234

GAC CCA AGT C-3’ which amplify a 73 bp fragment only from the parkin235

heterozygous control flies and the second set was 5’-CAA AGC CCT GTC CAA 236

AAT GC-3’ and 5’-GCG CGT GTG CAG ACC AT-3’; for MTF-1, the first primer 237

set was 5’-TGT CCG GCT GCG ATA AGG-3’ and 5’-GCC ATT GTG CAG ACG 238

AAG GT-3’ which amplify a 68 bp fragment from wild-type MTF-1 containing flies 239

and the second set was GCA TTC AAC ACG CGC TAC A-3’ and 5’-ACA GTT 240

GAA CGT CTC GCC ATT-3’; for MtnB, the first primer set was 5’-TTG CAA GGG 241

TTG TGG AAC AAA-3’ and 5’-TGC AGG CGC AGT TGT CC-3’ which amplify a 242

65 bp fragment and the second set was 5’-AAG TCG AGA AAT AGA TAC ATA 243

CAA GAT GGT-3’ and 5’-CGC ACT TTT GGG CCG AG-3’; for foi, the first primer 244

set was 5’-GTG GCT GCG GGT CTG TTC-3’ and 5’-TTT GTG CGA GGC CGA 245

GAT-3’ which amplify a 69 bp fragment and the second set was 5’-TGG CGA TGC 246

CCT ACT TCA C-3’ and 5’-TGA TCA TCC CCC GCT CAT-3’. 247

248

Detection of ROS levels 249

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

11

Fresh dichlorofluorescin diacetate (2,7-DCFH-DA) from Invitrogen – Molecular 250

Probes Cat # C369 was mixed with DMSO to make a 1 mM stock solution (=2,7-251

DCFH-DA/DMSO). A 40 μM working solution was prepared in HEPES buffer (30 252

mM). 50 heads of frozen adult flies of a specific genotype (same age) were removed 253

and collected in an Eppendorf tube. This was done in triplicate for each condition. 254

Each sample was then homogenized using cold protein homogenization buffer (1:3 255

w/v of 0.32 mM sucrose, 20 mM HEPES, 1 mM MgCl2, 0.5 mM PMSF protease 256

inhibitor at pH 7.4) and centrifuged for 20 minutes at 4oC and 20,000 g. Protein 257

content of the supernatant was determined using the Bio Rad diagnostics kit and a 258

final concentration of 0.4 mg/ml was used as the standard for the ROS assay of each 259

genotype tested. The fluorescence intensity (emission acquisition) was monitored for 260

45 minutes after the sample (20 μl) was kept in a cuvette (1.5 x 1.5 nm) in the 261

fluorimeter immediately after the addition of the DCFH-DA dye (2 μl) to prevent loss 262

of signal due to fading of fluorescence. Excitation of dye was at 485 nm and emission 263

at 520 nm. The curve area of fluorescence intensity which was recorded every 5 264

minutes (for 45 minutes) in the range of 500-600 nm was integrated and the total area 265

was used for comparison, with the final result obtained in counts per second. The 266

fluorimeter was standardized using 0.05% H2O2, a positive ROS generating species. 267

268

Dissection of ovaries 269

Ovaries from female parents were dissected in Grace’s insect medium (1X), GIBCO, 270

Invitrogen at room temperature (RT). The dissected ovaries were immediately fixed in 271

4% paraformaldehyde, 0.2% Triton-X-100 dissolved in Grace’s medium for 20 272

minutes without shaking, The fixative was washed three times with phosphate buffer 273

saline (PBS)+0.5% Triton (PBST) for 10 minutes each. The samples were then 274

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

12

labeled for one hour with fluorescently labeled phalloidin (Phalloidin-alexa 568, 275

molecular probes), diluted 1:200 in PBST at RT. This was followed by three washes 276

with PBST for 10 minutes each and a second labeling with Toto (nuclear stain) 277

diluted 1:1000 in PBST at RT. The samples were then washed three times with PBST 278

for 10 minutes each, followed by two washes with PBS for 10 minutes each and then 279

embedded in vectashield (mounting medium) overnight at -20oC. Ovarioles were 280

dissected from the ovaries and mounted on glass slides. Pictures were taken using the 281

confocal at 20X magnification. 282

283

Quantification of metal content 284

Female flies (park25/+, tub-MTF-1, park25/+ and yw and MTF-1 knockout controls) 285

were allowed to lay eggs on normal food or metal-supplemented food (100 μM 286

cadmium sulphate/ 500 μM copper sulphate/ 500 μM ammonium ferric citrate/ 4 mM 287

zinc chloride) for four days and removed afterwards. The resulting progeny were 288

collected at regular intervals and frozen. This procedure was repeated until the 289

required number of 50 flies was obtained in triplicate for each genotype. Each sample 290

set of frozen flies was then subjected to homogenization using cold protein 291

homogenization buffer (0.32 mM sucrose, 20 mM HEPES, 1 mM MgCl2, 0.5 mM 292

PMSF protease inhibitor at pH 7.4) and the samples were normalized for protein 293

content. A final concentration of 1 mg/ml was prepared by diluting the samples in 294

0.2M HNO3 to obtain a total assay volume of 1 ml. A highly sensitivity flame atomic 295

absorption spectrophotometer (FAAS; GTA-120/PSD-120, Varian Australia Pty Ltd, 296

Mulgrave, VIC, Australia) was used to detect the metal content in each genotype 297

assessed. Cd and Zn concentrations were recorded by the same flame. Likewise Cu 298

and Fe concentrations were measured together. 299

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

13

300

Muscle section and TEM 301

Dissected thoraces of two-day old anesthetized adult flies (park25/+, park25/25 and Act-302

Gal4; UAS-MTF-1,park25/25) were kept in ice-cold fixative (2.5% glutaraldehyde/0.1 303

M sodium cacodylate buffer adjusted to 328 mOsm/l with sucrose, pH 7.4) for 4 hours 304

at 4°C. Postfixation was performed with 1% OsO4 and 0.1 M sodium cacodylate, pH 305

7.4 for 2 hours at 4°C and the sample was washed overnight with 0.1 M sodium 306

cacodylate, pH 7.4 before going through a series of progressive dehydration steps in a 307

graded ethanol series of 70/80/96% alcohol for 10 minutes each, followed by three 10 308

min washes in 100% alcohol and a final 20 min wash in propylene oxide. The sample 309

was treated with a 1:1 propylene oxide: Epon (Epon 812) mix for 2 hours and 310

embedded in Epon overnight at 70oC. Blocks of thoraces were trimmed and semi-thin 311

sections of dorsal longitudinal muscles were stained with Toluidine Blue dye, which 312

labels nucleic acids, hence staining both nuclei and cytoplasm (5). Ultrathin sections 313

of 70 nm thickness were made with an ultramicrotome for the selected sections. For 314

TEM, sections were contrast-stained on the grid first with 2% uranyl acetate, then 315

with 2.5% lead citrate (Reynold’s), each of them for twenty minutes at room 316

temperature. The sections were inspected with a Philips CM100 TEM with GATAN 317

Orius Camera. 318

319

Statistical Analysis 320

JMP software (SAS Institute) was used for statistical evaluations. Lifespan (survival) 321

assays were analyzed with the Kaplan–Meier log-rank statistical test. Brain ROS 322

levels and qPCR results were compared by one-way ANOVA. Results are expressed 323

as mean ± standard deviation. 324

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

14

325

Results 326

327

Synthetic lethality of combined parkin and MTF-1 mutants 328

329

The metal-responsive transcription factor (MTF-1) is a key regulator of metal 330

homeostasis in Drosophila (17, 60, 75). Based on our findings of a connection 331

between the parkin knockout and trace metal status (55), we tested the effect of an 332

MTF-1 knockout in a parkin mutant background. The result was clear cut, in that no 333

surviving double mutant was ever observed in 50 independent crosses. This “synthetic 334

lethality,” observed at the pupal stage, was rescued by a cDNA transgene of MTF-1335

driven by the constitutive tubulin enhancer/promoter. In a genetic cross between 336

parents heterozygous for the parkin and MTF-1 recombined deletions (Figure 1), 69 337

out of a total of 217 progeny (32%) were parkin and MTF-1 homozygous knockouts 338

also expressing an MTF-1 transgene (statistical expectation, 40%). This rescue by an 339

MTF-1 cDNA transgene confirms the absence of any secondary hits as the cause of 340

the observed lethality. Furthermore, it also excludes the possibility that an intronic 341

open reading frame located within the MTF-1 gene (D. Steiger, K. Steiner and WS, 342

unpublished) is responsible for the effect and not MTF-1 itself. To find out what 343

condition could overcome the pupal lethality, we maintained the heterozygous parkin, 344

MTF-1 parent flies on N-acetylcysteine (NAC), which is a precursor to glutathione, an 345

established antioxidant. Since either condition, lack of a functional parkin gene or of 346

an MTF-1 gene, increases reactive oxygen species (ROS) (see below), we reasoned 347

that keeping a lower ROS level by other means might also overcome the synthetic 348

lethality of the double mutants. After testing different concentrations of NAC an 349

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

15

optimal supplement of 15 mM was chosen for the main experiment. Indeed, raising 350

the progeny under this condition resulted in a substantial rescue of the synthetic 351

lethality: of the total progeny, 19% was homozygous parkin and MTF-1 double 352

mutant (statistical expectation, 25%) (Table 1A). Other antioxidants tested included 353

ascorbate, zinc, and metal chelators of copper and iron (BCS and BPS, respectively) 354

which we had shown before to positively influence the parkin mutant phenotype (55, 355

56). However, none of these was able to rescue synthetic lethality. 356

357

MTF-1 overexpression rescues the lifespan and the low eclosing frequency of 358

parkin mutants 359

360

We examined the lifespan of Drosophila MTF-1-overexpressing transgenic lines (tub-361

MTF-1 and Act-Gal4; UAS-MTF-1) in a park25/25 background. These experiments 362

illustrated that an elevated expression of MTF-1 from the ubiquitously active tubulin363

enhancer/promoter prolonged the lifespan of parkin mutants significantly, from a 364

median of 7 days for the mutants alone to 21 days (Figure 2A). In this experiment, the 365

maximal lifespan was extended from 12 to 41 days by the MTF-1 transgene (Figure 366

2A). The stronger combination with actin-Gal4 driving UAS-MTF-1 revealed a 367

similar effect: in an experiment with 23 independent replicas, 10% of the mutant 368

animals were still alive at day 34 (Figure 2B). Overexpression of MTF-1 in control 369

and wild type flies (park25/+ or park+/+) did not increase their normal lifespan (data 370

not shown). Also, in an independent study, MTF-1 overexpression did not extend 371

lifespan of flies kept on standard food (4). Elevated MTF-1 expression not only 372

prolonged the lifespan of parkin mutant adult flies but also enhanced survival during 373

development. In a genetic cross involving parkin heterozygous parents, only 2.5% of 374

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

16

the eclosing progeny flies were homozygous parkin mutants (statistical expectation, 375

25%). In comparison, the same cross but also expressing an MTF-1 transgene resulted 376

in a 15% eclosure frequency (Table IB). 377

378

Elevated expression of MTF-1 rescues female fertility and fecundity of parkin 379

mutant flies 380

381

Strikingly, female fertility and fecundity was completely rescued by MTF-1. When 382

crossed with wild type males, parkin mutant females with the tubulin-driven MTF-1383

transgene produced the same number of progeny as a cross of wild type males and 384

females. Drosophila gonad formation requires a complex morphogenetic process (35, 385

37). As in the majority of metazoans, Drosophila oogenesis occurs within the ovarian 386

follicles in which germline cells develop in close proximity to specialized somatic 387

cells (Figure 3A-A’’). Parkin mutant females lack the proper spatio-temporal 388

development in the germarium and thus have stunted ovaries with few mature 389

oocytes, which fail to get fertilized (Figure 3B-B’’’). The restoration of female 390

fertility by strong MTF-1 expression was also evident at the morphological level: 391

dissected ovaries showed a normalized structure with follicles formed in the 392

germarium and mature stages in the posterior regions of the ovariole, with several 393

oocytes ready for fertilization (Figure 3C-C’’). In contrast, the sterility phenotype of 394

parkin mutant males which is due to defective spermatogenesis at the 395

individualization step (24) was not rescued. This suggests that Parkin is particularly 396

important for male fertility. 397

398

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

17

Improved locomotion and rescued mitochondrial/myofibrillar morphology of 399

parkin mutants with enhanced MTF-1 expression 400

401

Strong MTF-1 expression dramatically improved the climbing ability of parkin402

mutant flies (Figure 4). What is more, they generally moved around fast, responded 403

by running away when physically perturbed, jumped and occasionally displayed short 404

flight episodes (data not shown). To investigate the effect of elevated MTF-1 405

expression on muscle morphology we examined the ultrastructure of the indirect 406

flight muscle in heterozygous control flies, parkin mutants and the MTF-1 transgenic 407

flies. Cross-thoracic sections of control adults analyzed by transmission electron 408

microscopy (TEM) revealed well organized muscle fibres in parallel stripes with a 409

regular M- and Z-line banding pattern and darkly stained, electron-dense 410

mitochondria with regularly packed cristae (Figure 5A-C). In contrast, age-matched 411

parkin mutants had abnormal muscle structure with large vacuoles, a reduced muscle 412

content with mostly irregular arrangement and enlarged mitochondria with 413

disintegrated cristae (Figure 5D-F). The MTF-1 overexpressing parkin mutant flies 414

displayed a clear rescue effect in that muscle fibre structure was more regular with 415

less prominent vacuoles; moreover, mitochondria had more densely packed cristae 416

with considerably lesser signs of disintegration in comparison to parkin mutants 417

(Figure 5G-I). 418

419

MTF-1 dependent expression of metallothioneins is higher in parkin mutant 420

Drosophila421

422

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

18

Owing to their high cysteine content, metallothioneins can act as antioxidants, in 423

addition to their obvious role as metal chelators (33, 38). Basal and induced levels of 424

metallothionein expression depend on the transcription factor MTF-1 (17, 51, 74). To 425

find out whether metallothionein genes could be induced by MTF-1 in a parkin 426

mutant background, we used a reporter line in which a yellow fluorescent protein 427

(EYFP) is driven by the promoter of MtnA, the most highly expressed Drosophila428

metallothionein gene. Compared to heterozygous controls, even in the absence of any 429

heavy metal load the basal expression of this reporter was increased, likely due to 430

elevated oxidative stress in the parkin mutant flies (Figure 6C and D). This metal-431

independent upregulation of the MtnA promoter, was strictly dependent on MTF-1, 432

since no trace of fluorescence could be detected in flies lacking MTF-1 (data not 433

shown). RT-PCR of parkin mutants also revealed elevated transcript levels of MTF-1434

(2-fold) (Figure 7A) and the embryo-enriched metallothionein MtnB (6-fold) (Figure 435

7B) compared to park25/+ flies. MTF-1 overexpression in the park25/25 flies was 436

achieved from the tubulin promoter or indirectly with the stronger UAS-Act-Gal4 437

system, both induced a more than 200-fold increase in MtnB transcripts (Figure 7B). 438

Conversely, the level of parkin transcripts was increased in the MTF-1 knockout flies 439

(Figure 7C). This pattern of regulation can be explained by a partial redundancy of 440

Parkin and MTF-1 where one is upregulated to compensate for the loss of the other. 441

The synthetic lethality of a combined knockout of parkin and MTF-1 genes mentioned 442

above is in line with this hypothesis. Another experiment revealed that the transcript 443

levels of the zinc importer foi (37) in parkin mutants were enhanced 3-fold by 444

elevated MTF-1 expression, which may contribute to the normalized structure of 445

ovaries and rescued fertility of female parkin mutants (Figure 7D). This hypothesis is 446

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

19

further supported by the increased level of zinc ions found in tub-MTF-1; park25/25 447

flies (discussed below, Figure 8B). 448

449

Elevated MTF-1 expression confers resistance to oxidative stress and restores 450

metal homeostasis 451

452

Parkin mutant flies display high levels of ROS indicative of intrinsic oxidative stress 453

(23, 72). In agreement with MTF-1 having an antioxidant function, we observed a 454

substantial decrease of ROS levels in the heads of parkin mutants expressing an MTF-455

1 transgene: ROS dropped to approximately half of the levels in park25/25 flies (Figure 456

8A). Previously we had observed that limiting the availability of redox-active metals, 457

achieved by supplementing the food with chelators for copper and iron (BCS and 458

BPS, respectively), also increased the lifespan of parkin mutant flies (55). 459

Furthermore, the w;tub-MTF1;park25/25 flies raised on the metal-chelator-460

supplemented food displayed a somewhat lower ROS level (Figure 8A). ROS levels 461

were not significantly changed in heterozygous control flies, either upon ubiquitous 462

MTF-1 overexpression or following dietary intake of metal-chelators. The MTF-1463

knockout flies showed the highest levels of ROS, probably due to the reduced 464

expression of MTF-1-dependent antioxidant genes such as metallothioneins (Figure 465

8A). Metals like zinc and the redox active copper and iron are required in trace 466

amounts for several structural and biological processes in organisms (66). Park25/25467

flies display not only reduced basal levels of zinc (see also (56)) but also of copper 468

and iron in comparison to control flies (Figure 8B). Tubulin-driven MTF-1 expression 469

in park25/25 restores the basal level of these metals (Figure 8B). Concentrations of 470

cadmium are generally low since it is a non-essential, toxic metal. Upon 471

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

20

supplementing fly food with metals, their levels became quite similar in all three 472

genotypes tested (Figure 8C). This is particularly important in the case of zinc 473

supplementation, which we had previously shown to improve the condition of parkin474

mutant Drosophila (56). 475

476

Human MTF-1 or elevated metallothionein expression also improve condition of 477

parkin mutants478

479

Human MTF-1 has been expressed in Drosophila and shown to largely, but not 480

completely, rescue the metal sensitivity of Drosophila lacking its endogenous MTF-1 481

(6). We therefore tested the effect of actin-Gal4-driven hMTF-1 expression in a 482

parkin mutant background. Indeed the lifespan of parkin mutants was increased from 483

a median of 7 days to 19 days, which is close to the 21 days obtained with elevated 484

Drosophila-MTF-1 expression (Figure 9). Other rescue effects paralleled those 485

observed with elevated Drosophila MTF-1 expression (see above) but were less 486

pronounced (data not shown). 487

488

Metallothionein genes are the major targets of MTF-1; in Drosophila, metallothionein489

A (MtnA) shows the strongest expression. Thus, we also tested if overexpression of 490

MtnA in a parkin mutant background exerted a similar beneficial effect as MTF-1491

overexpression. To this end we crossed inter se three independent lines with tubulin-492

driven MtnA overexpression in a parkin heterozygous background to raise parkin493

homozygous knockouts. The median lifespan of the parkin mutants was extended up 494

to 17 days (Figure 9) but other rescue effects associated with elevated MTF-1 495

expression (discussed in results) were not observed. 496

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

21

497

Discussion 498

499

Here we show that metal responsive transcription factor (MTF-1) plays a crucial role 500

in modulating the severity of a Parkin loss-of-function phenotype. On the one hand, 501

the combined loss of Parkin and MTF-1 is not viable, i.e., displays synthetic lethality. 502

On the other hand, elevated expression of MTF-1 dramatically improves the condition 503

of parkin mutant flies: there is an overall extension of life span, females regain 504

normal fertility, and the motoric abilities of flies improve to the point that they can 505

walk fast and even display short episodes of flight. The latter is noteworthy since 506

flight muscles have a high energy consumption that depends on robust mitochondrial 507

function. At the histological level, the degenerated mitochondria characteristically 508

seen in parkin mutants are rescued to a more regular, electron-dense structure 509

following MTF-1 overexpression. Additionally, flight muscles have an improved 510

myofibrillar arrangement. In earlier studies, mitochondrial and muscle degeneration 511

observed in parkin mutant flies were proposed to be a result of excessive oxidative 512

stress (24, 48). Mitochondrial malfunction can indeed result in an increased 513

susceptibility to oxygen radical damage, and mitochondria-associated increase in ROS 514

production has been implicated in Parkinson’s disease (19, 26, 73). In line with this 515

concept, the major target genes of MTF-1 are metallothioneins (MTs), which encode 516

small, cysteine-rich proteins that can scavenge heavy metals, notably the redox-active 517

copper, and ROS. The elevated basal level of MTF-1 and MT transcripts in parkin518

mutant flies can thus be seen as a compensatory attempt to counteract enhanced ROS 519

levels (24). In accordance with such a scenario is the rescue effect seen upon food 520

supplementation with the antioxidant N-acetylcysteine, which however falls short of 521

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

22

the dramatic effect seen with elevated MTF-1 expression. Besides metallothioneins 522

and a number of other stress-associated genes, ferritin genes are a major target of 523

MTF-1 in Drosophila (74). Ferritins are well-characterized iron binding proteins 524

which keep iron in a soluble and non-toxic form in the cell. Thus ROS production by 525

redox-active iron might be lowered via upregulation of ferritin levels. The multiple 526

targets of MTF-1 help to explain why overexpression of metallothionein alone was 527

less effective than MTF-1 overexpression in improving the condition of parkin528

mutants. In a separate series of experiments we found that strong expression of an 529

ortholog, the human MTF-1, also increased lifespan, rescued female fertility and 530

improved locomotion ability, although the effects were less pronounced than with 531

Drosophila’s own MTF-1, presumably due to the evolutionary distance between 532

mammals and insects. This is in line with previous findings from our laboratory that 533

mammalian and Drosophila MTF-1 transgenes are able to largely, but not completely, 534

compensate for each other’s absence (6). 535

536

Although we observed remarkable improvements following elevated MTF-1 537

expression, a complete rescue of parkin mutants, including male fertility and 538

sustained flight ability, was only observed with a parkin transgene. Furthermore, it 539

has to be pointed out that although MTF-1 knockout flies display high ROS levels, 540

they show no signs of Parkinson’s disease-like symptoms. Together these findings are 541

consistent with the idea that oxidative stress is an important but not the sole culprit in 542

PD etiology (1, 14, 49). Nevertheless, the dramatic effect of MTF-1 on a parkin loss-543

of-function mutation underscores the importance of this transcriptional regulator in 544

cellular stress response. 545

546

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

23

Acknowledgments 547

548

We are indebted to Drs. Jessica Greene and Leo Pallanck (University of Washington) 549

for providing the park25/25 flies, to Gery Barmettler and Theresa Bruggmann (Center 550

for Microscopy and Image analysis) for TEM analysis of muscle sections, to Martin 551

Moser for real-time analysis, Dr. Eva Freisinger and Tamara Huber (Inorganic 552

Chemistry, UZH) for FAAS experiments, Ivan Ostojic (FMI Basel) for statistical 553

analyses, and to Dr. Ben Schuler (Biochemistry, UZH) for providing the fluorimeter 554

for ROS analysis. We also thank Till Strassen for maintenance of fly stocks and Dr. 555

George Hausmann for critical reading of the manuscript. This work was supported by 556

the Swiss National Science Foundation grant 31003A-113993 and the University of 557

Zürich.558

559

Conflict of interest 560

561

The authors declare that they have no conflict of interest. 562

563

Figure Legends 564

565

Figure 1. Scheme of genetic cross to obtain strong MTF-1 expression in double 566

mutant background. An MTF-1 cDNA transgene driven by the tubulin promoter 567

was combined with homozygous null mutations of both MTF-1 (MTF-1-) (17) and 568

parkin (park25) (24); progeny in red. The Sp marker on the second chromosome gives 569

a uniform-length side-bristle phenotype and the CyO second chromosome balancer 570

results in curly wings. TM3 and TM6B are third chromosome balancers which display 571

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

24

a serrated wing phenotype and a dense side bristle phenotype, respectively. Balancers 572

are lethal in homozygous form; progeny marked with a cross indicate lethality of that 573

particular genotype. Chromosome 1 is wild type. 574

575

Figure 2. Enhanced lifespan of parkin mutants expressing an MTF-1 transgene.576

(A) A cDNA transgene of Drosophila-MTF-1 driven by the ubiquitously active 577

tubulin enhancer/promoter prolongs the lifespan of parkin mutants (park25/25) up to 41 578

days. For survival of park25/25 vs. tub-MTF-1, park25/25 flies, p<0.001. (B) Strong 579

expression of a UAS-MTF-1 transgene driven by actin-Gal4 in a parkin mutant 580

background also extends lifespan, as tested in multiple sets of Act-Gal4; UAS-MTF-581

1, park25/25 flies. In both (A) and (B), heterozygous park25/+ flies with MTF-1 582

overexpression had a similar lifespan as park25/+ controls (data not shown). 583

584

Figure 3. Elevated MTF-1 expression restores ovary structures and restores 585

fertility of parkin mutant females. (A-A’’) Normal ovariole structures of fertile 586

control female flies (park25/+). (B-B’’’) Infertile female parkin mutants (park25/25)587

have a distorted ovary structure with very few mature eggs. (C-C’’) Upon expression 588

of a tubulin-driven MTF-1 transgene ovary structures are normalised, resulting in 589

normal fertility. a, anterior with germarium; p, posterior with vitellarium; broken 590

arrows indicate mature eggs; full arrows developing stages of eggs. Phalloidin (red) 591

stains tubulin structures and Toto (blue) stains the nuclei.592

593

Figure 4. MTF-1 transgene expression restores the climbing ability of parkin 594

mutants. Tubulin enhancer/promoter-driven MTF-1 expression largely rescues the 595

locomotion ability of park25/25 flies. In control flies, MTF-1 overexpression does not 596

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

25

further improve climbing ability. Data shown represents mean value ± standard 597

deviation of each group tested every other day up to 8 days. Asterisks show highly 598

significant differences between parkin control (park25/+) and parkin mutant (park25/25)599

flies on each day of the assay (p< 0.0001). 600

601

Figure 5. Strong MTF-1 expression improves muscle and mitochondrial 602

morphology of parkin mutants. (A-C) Transverse sections of indirect flight muscles 603

(IFMs) show well preserved muscle in park25/+ heterozygous controls with a regular 604

myofibril arrangement (white arrows) and many electron-dense mitochondria (broken 605

red arrows). (D-F) park25/25 adult IFMs show an irregular myofibrillar arrangement 606

with diffuse Z-lines and M-bands and numerous vacuoles. Mitochondria are swollen 607

with fragmented cristae (red arrows). (G-I) Myofibril and mitochondrial integrity of 608

parkin mutants is restored by MTF-1 overexpression. Mitochondria are more dense 609

(broken white arrows) and muscle structure is more regular (broken green arrows), 610

although occasional vacuoles are observed (green arrows). The scale bar is for top and 611

middle panels while the bottom panel is at a higher magnification, shown for clarity. 612

613

Figure 6. MTF-1 activity is upregulated in a parkin deficient background. Top: 614

Transgenic MtnA/EYFP reporter gene (6). Bottom: (A-B) MtnA-EYFP, park25/+ and 615

(C-D) MtnA-EYFP, park25/25. Pictures of 1-2 day old adult flies were taken at 730 ms 616

exposure with a Leica fluorescence microscope. ‘a’ and ‘p’ show anterior and 617

posterior ends, respectively. 618

619

Figure 7. Increased MTF-1 and metallothionein (MtnB) transcript levels in 620

parkin mutants that also express an MTF-1 transgene. Real-time transcript-levels 621

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

26

of (A) MTF-1, (B) MtnB, (C) parkin and (D) foi in park25/+ and park25/25 and w; tub-622

MTF-1, park25/25 and Act-Gal4; UAS-MTF-1; park25/+ and Act-Gal4; UAS-MTF-1; 623

park25/25 flies with MTF-1 knockout flies as an appropriate control. All flies were 624

raised on normal food (NF). MTF-1 null flies show no MtnB and parkin null flies 625

show no parkin transcripts. (B) MTF-1 overexpression from the tubulin 626

enhancer/promoter or via the UAS-Act-Gal4 system induced a 234-fold and 270-fold 627

increase in MtnB transcripts, respectively. *** indicates significant difference 628

between parkin mutant adult flies and parkin mutants with elevated MTF-1 629

expression (p<0.0001). 630

631

Figure 8. MTF-1 reduces reactive oxygen species (ROS) levels in parkin mutants 632

and restores metal homeostasis. (A) park25/25 flies (NF) show high amounts of ROS. 633

MTF-1 transgene expression, or treatment with chelators of redox-active metals (BPS 634

and BCS) reduce ROS levels. parkin heterozygous controls (park25/+) do not show 635

significant differences in ROS levels with or without MTF-1-overexpression or 636

dietary supplementation with metal chelators. The MTF-1 knockout shows the highest 637

ROS level. ***, p<0.001 and **, p<0.01, chelator-supplemented compared to normal 638

food for the same genotype (black stars), or compared to the same treatment between 639

genotypes (red stars). (B) Elevated MTF-1 expression in a parkin mutant background 640

restores normal basal levels of the essential trace metals copper, iron and zinc when 641

flies are raised on NF (normal food). (C) Differences in metal content between 642

controls, park25/25 and tub-MTF-1, park25/25 flies are largely leveled out by metal 643

supplementation of the food. 644

645

Figure 9. Strong expression of human MTF-1 or of Drosophila metallothionein 646

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

27

prolongs lifespan of parkin mutants. A human-MTF-1 transgene driven by actin-647

Gal4 extends the median lifespan of parkin mutant flies from 7 (red) to 19 days (light 648

blue). Direct overexpression of metallothionein MtnA by tubulin promoter also 649

enhances the median lifespan of parkin mutants up to 17 days (green). An even better 650

extension up to a median of 21 days is observed with a tubulin-driven trangene of 651

Drosophila MTF-1 (dark blue). park25/+ (black) and park25/25 (red) serve as controls. 652

653

Table I. (A) Partial rescue of synthetic lethality of combined parkin and MTF-1654

mutants by N-acetylcysteine: MTF-1, parkin heterozygous parents were crossed 655

inter se on food supplemented with 15mM NAC. (B) Increased frequency of flies 656

reaching adulthood among parkin mutants overexpressing MTF-1. park25/25657

mutants were obtained from parkin heterozygous parents crossed inter se. In both (A) 658

and (B), egg laying was allowed for two days by equal numbers of parents and 659

eclosing progeny flies were counted thereafter. Three independent crosses were done. 660

661

References 662

663

1. Abbott, R. D., G. W. Ross, L. R. White, W. T. Sanderson, C. M. Burchfiel, 664M. Kashon, D. S. Sharp, K. H. Masaki, J. D. Curb, and H. Petrovitch.6652003. Environmental, life-style, and physical precursors of clinical Parkinson's 666disease: recent findings from the Honolulu-Asia Aging Study. J Neurol 250667Suppl 3:III30-9. 668

2. Andrews, G. K. 2000. Regulation of metallothionein gene expression by 669oxidative stress and metal ions. Biochem Pharmacol 59:95-104.670

3. Auf der Maur, A., T. Belser, G. Elgar, O. Georgiev, and W. Schaffner.6711999. Characterization of the transcription factor MTF-1 from the Japanese 672pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal 673stress response. Biol Chem 380:175-85. 674

4. Bahadorani, S., S. Mukai, D. Egli, and A. J. Hilliker. 2010. Overexpression 675of metal-responsive transcription factor (MTF-1) in Drosophila melanogaster 676ameliorates life-span reductions associated with oxidative stress and metal 677toxicity. Neurobiol Aging 31:1215-26.678

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

28

5. Balabanova, M., L. Popova, and R. Tchipeva. 2003. Dyes in dermatology. 679Clin Dermatol 21:2-6.680

6. Balamurugan, K., D. Egli, A. Selvaraj, B. Zhang, O. Georgiev, and W. 681Schaffner. 2004. Metal-responsive transcription factor (MTF-1) and heavy 682metal stress response in Drosophila and mammalian cells: a functional 683comparison. Biol Chem 385:597-603. 684

7. Brugnera, E., O. Georgiev, F. Radtke, R. Heuchel, E. Baker, G. R. 685Sutherland, and W. Schaffner. 1994. Cloning, chromosomal mapping and 686characterization of the human metal-regulatory transcription factor MTF-1. 687Nucleic Acids Res 22:3167-73. 688

8. Bueler, H. 2010. Mitochondrial dynamics, cell death and the pathogenesis of 689Parkinson's disease. Apoptosis. 690

9. Chen, W. Y., J. A. John, C. H. Lin, and C. Y. Chang. 2002. Molecular 691cloning and developmental expression of zinc finger transcription factor MTF-6921 gene in zebrafish, Danio rerio. Biochem Biophys Res Commun 291:798-693805.694

10. Cookson, M. R., G. Xiromerisiou, and A. Singleton. 2005. How genetics 695research in Parkinson's disease is enhancing understanding of the common 696idiopathic forms of the disease. Curr Opin Neurol 18:706-11. 697

11. Culotta, V. C., M. Yang, and T. V. O'Halloran. 2006. Activation of 698superoxide dismutases: putting the metal to the pedal. Biochim Biophys Acta 6991763:747-58. 700

12. Dalton, T. P., Q. Li, D. Bittel, L. Liang, and G. K. Andrews. 1996. 701Oxidative stress activates metal-responsive transcription factor-1 binding 702activity. Occupancy in vivo of metal response elements in the metallothionein-703I gene promoter. J Biol Chem 271:26233-41.704

13. Dawson, T. M., and V. L. Dawson. 2003. Molecular pathways of 705neurodegeneration in Parkinson's disease. Science 302:819-22.706

14. Dexter, D. T., C. J. Carter, F. R. Wells, F. Javoy-Agid, Y. Agid, A. Lees, 707P. Jenner, and C. D. Marsden. 1989. Basal lipid peroxidation in substantia 708nigra is increased in Parkinson's disease. J Neurochem 52:381-9. 709

15. Ebadi, M., H. Brown-Borg, H. El Refaey, B. B. Singh, S. Garrett, S. 710Shavali, and S. K. Sharma. 2005. Metallothionein-mediated neuroprotection 711in genetically engineered mouse models of Parkinson's disease. Brain Res Mol 712Brain Res 134:67-75. 713

16. Egli, D., J. Domenech, A. Selvaraj, K. Balamurugan, H. Hua, M. 714Capdevila, O. Georgiev, W. Schaffner, and S. Atrian. 2006b. The four 715members of the Drosophila metallothionein family exhibit distinct yet 716overlapping roles in heavy metal homeostasis and detoxification. Genes Cells 71711:647-58. 718

17. Egli, D., A. Selvaraj, H. Yepiskoposyan, B. Zhang, E. Hafen, O. Georgiev, 719and W. Schaffner. 2003. Knockout of 'metal-responsive transcription factor' 720MTF-1 in Drosophila by homologous recombination reveals its central role in 721heavy metal homeostasis. Embo J 22:100-8. 722

18. Egli, D., H. Yepiskoposyan, A. Selvaraj, K. Balamurugan, R. Rajaram, A. 723Simons, G. Multhaup, S. Mettler, A. Vardanyan, O. Georgiev, and W. 724Schaffner. 2006a. A family knockout of all four Drosophila metallothioneins 725reveals a central role in copper homeostasis and detoxification. Mol Cell Biol 72626:2286-96. 727

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

29

19. Gandhi, S., A. Wood-Kaczmar, Z. Yao, H. Plun-Favreau, E. Deas, K. 728Klupsch, J. Downward, D. S. Latchman, S. J. Tabrizi, N. W. Wood, M. R. 729Duchen, and A. Y. Abramov. 2009. PINK1-associated Parkinson's disease is 730caused by neuronal vulnerability to calcium-induced cell death. Mol Cell 73133:627-38. 732

20. Gasser, T. 2005. Genetics of Parkinson's disease. Curr Opin Neurol 18:363-9.73321. Ghoshal, K., and S. Jacob. 2000. Regulation of metallothionein gene 734

expression. Progress in Nucleic Acid Research and Molecular Biology 73566:357-384. 736

22. Ghoshal, K., S. Majumder, Q. Zhu, J. Hunzeker, J. Datta, M. Shah, J. F. 737Sheridan, and S. T. Jacob. 2001. Influenza virus infection induces 738metallothionein gene expression in the mouse liver and lung by overlapping 739but distinct molecular mechanisms. Mol Cell Biol 21:8301-17.740

23. Greene, J. C., A. J. Whitworth, L. A. Andrews, T. J. Parker, and L. J. 741Pallanck. 2005. Genetic and genomic studies of Drosophila parkin mutants 742implicate oxidative stress and innate immune responses in pathogenesis. Hum 743Mol Genet 14:799-811. 744

24. Greene, J. C., A. J. Whitworth, I. Kuo, L. A. Andrews, M. B. Feany, and 745L. J. Pallanck. 2003. Mitochondrial pathology and apoptotic muscle 746degeneration in Drosophila parkin mutants. Proc Natl Acad Sci U S A 747100:4078-83. 748

25. Gunes, C., R. Heuchel, O. Georgiev, K. H. Muller, P. Lichtlen, H. 749Bluthmann, S. Marino, A. Aguzzi, and W. Schaffner. 1998. Embryonic 750lethality and liver degeneration in mice lacking the metal-responsive 751transcriptional activator MTF-1. Embo J 17:2846-54. 752

26. Haque, M. E., K. J. Thomas, C. D'Souza, S. Callaghan, T. Kitada, R. S. 753Slack, P. Fraser, M. R. Cookson, A. Tandon, and D. S. Park. 2008. 754Cytoplasmic Pink1 activity protects neurons from dopaminergic neurotoxin 755MPTP. Proc Natl Acad Sci U S A 105:1716-21. 756

27. Heuchel, R., F. Radtke, O. Georgiev, G. Stark, M. Aguet, and W. 757Schaffner. 1994. The transcription factor MTF-1 is essential for basal and 758heavy metal-induced metallothionein gene expression. Embo J 13:2870-5. 759

28. Jenner, P., and C. W. Olanow. 1996. Oxidative stress and the pathogenesis 760of Parkinson's disease. Neurology 47:S161-70. 761

29. Kagi, J. H. 1991. Overview of metallothionein. Methods Enzymol 205:613-76226.763

30. Klaassen, C. D., J. Liu, and S. Choudhuri. 1999. Metallothionein: an 764intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol 765Toxicol 39:267-94. 766

31. La Fontaine, S., and J. F. Mercer. 2007. Trafficking of the copper-ATPases, 767ATP7A and ATP7B: role in copper homeostasis. Arch Biochem Biophys 768463:149-67. 769

32. Langmade, S. J., R. Ravindra, P. J. Daniels, and G. K. Andrews. 2000. 770The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 771gene. J Biol Chem 275:34803-9.772

33. Levy, M. A., Y. H. Tsai, A. Reaume, and T. M. Bray. 2001. Cellular 773response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice 774exposed to hyperoxia. Am J Physiol Lung Cell Mol Physiol 281:L172-82. 775

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

30

34. Lichtlen, P., and W. Schaffner. 2001. Putting its fingers on stressful 776situations: the heavy metal-regulatory transcription factor MTF-1. Bioessays 77723:1010-7. 778

35. Lin, H. 1998. The self-renewing mechanism of stem cells in the germline. 779Curr Opin Cell Biol 10:687-93. 780

36. Lindert, U., M. Cramer, M. Meuli, O. Georgiev, and W. Schaffner. 2009. 781Metal-responsive transcription factor 1 (MTF-1) activity is regulated by a 782nonconventional nuclear localization signal and a metal-responsive 783transactivation domain. Mol Cell Biol 29:6283-93. 784

37. Mathews, W. R., F. Wang, D. J. Eide, and M. Van Doren. 2005. 785Drosophila fear of intimacy encodes a Zrt/IRT-like protein (ZIP) family zinc 786transporter functionally related to mammalian ZIP proteins. J Biol Chem 787280:787-95. 788

38. Miura, T., S. Muraoka, and T. Ogiso. 1997. Antioxidant activity of 789metallothionein compared with reduced glutathione. Life Sci 60:PL 301-9. 790

39. Miyazaki, I., M. Asanuma, H. Hozumi, K. Miyoshi, and N. Sogawa. 2007. 791Protective effects of metallothionein against dopamine quinone-induced 792dopaminergic neurotoxicity. FEBS Lett 581:5003-8.793

40. Mokdad, R., A. Debec, and M. Wegnez. 1987. Metallothionein genes in 794Drosophila melanogaster constitute a dual system. Proc Natl Acad Sci U S A 79584:2658-62. 796

41. Murphy, B. J., G. K. Andrews, D. Bittel, D. J. Discher, J. McCue, C. J. 797Green, M. Yanovsky, A. Giaccia, R. M. Sutherland, K. R. Laderoute, and 798K. A. Webster. 1999. Activation of metallothionein gene expression by 799hypoxia involves metal response elements and metal transcription factor-1. 800Cancer Res 59:1315-22. 801

42. Nelson, N. 1999. Metal ion transporters and homeostasis. Embo J 18:4361-71. 80243. Olanow, C. W., and W. G. Tatton. 1999. Etiology and pathogenesis of 803

Parkinson's disease. Annu Rev Neurosci 22:123-44. 80444. Otsuka, F., I. Okugaito, M. Ohsawa, A. Iwamatsu, K. Suzuki, and S. 805

Koizumi. 2000. Novel responses of ZRF, a variant of human MTF-1, to in 806vivo treatment with heavy metals. Biochim Biophys Acta 1492:330-40. 807

45. Palacino, J. J., D. Sagi, M. S. Goldberg, S. Krauss, C. Motz, M. Wacker, 808J. Klose, and J. Shen. 2004. Mitochondrial dysfunction and oxidative damage 809in parkin-deficient mice. J Biol Chem 279:18614-22.810

46. Palmiter, R. D. 1998. The elusive function of metallothioneins. Proc Natl 811Acad Sci U S A 95:8428-30. 812

47. Pendleton, R. G., F. Parvez, M. Sayed, and R. Hillman. 2002. Effects of 813pharmacological agents upon a transgenic model of Parkinson's disease in 814Drosophila melanogaster. J Pharmacol Exp Ther 300:91-6.815

48. Pesah, Y., T. Pham, H. Burgess, B. Middlebrooks, P. Verstreken, Y. 816Zhou, M. Harding, H. Bellen, and G. Mardon. 2004. Drosophila parkin 817mutants have decreased mass and cell size and increased sensitivity to oxygen 818radical stress. Development 131:2183-94. 819

49. Petrovitch, H., G. W. Ross, R. D. Abbott, W. T. Sanderson, D. S. Sharp, 820C. M. Tanner, K. H. Masaki, P. L. Blanchette, J. S. Popper, D. Foley, L. 821Launer, and L. R. White. 2002. Plantation work and risk of Parkinson 822disease in a population-based longitudinal study. Arch Neurol 59:1787-92.823

50. Quaife, C. J., S. D. Findley, J. C. Erickson, G. J. Froelick, E. J. Kelly, B. 824P. Zambrowicz, and R. D. Palmiter. 1994. Induction of a new 825

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

31

metallothionein isoform (MT-IV) occurs during differentiation of stratified 826squamous epithelia. Biochemistry 33:7250-9. 827

51. Radtke, F., R. Heuchel, O. Georgiev, M. Hergersberg, M. Gariglio, Z. 828Dembic, and W. Schaffner. 1993. Cloned transcription factor MTF-1 829activates the mouse metallothionein I promoter. Embo J 12:1355-62. 830

52. Riparbelli, M. G., and G. Callaini. 2007. The Drosophila parkin homologue 831is required for normal mitochondrial dynamics during spermiogenesis. Dev 832Biol 303:108-20.833

53. Rossi, L., M. Arciello, C. Capo, and G. Rotilio. 2006. Copper imbalance and 834oxidative stress in neurodegeneration. Ital J Biochem 55:212-21. 835

54. Rouault, T. A., and S. Cooperman. 2006. Brain iron metabolism. Semin 836Pediatr Neurol 13:142-8.837

55. Saini, N., S. Oelhafen, H. Hua, O. Georgiev, W. Schaffner, and H. Bueler.8382010. Extended lifespan of Drosophila parkin mutants through sequestration 839of redox-active metals and enhancement of anti-oxidative pathways. 840Neurobiol Dis 40:82-92.841

56. Saini, N., and W. Schaffner. 2010. Zinc supplement greatly improves the 842condition of parkin mutant Drosophila. Biol Chem 391:513-8.843

57. Sang, T. K., H. Y. Chang, G. M. Lawless, A. Ratnaparkhi, L. Mee, L. C. 844Ackerson, N. T. Maidment, D. E. Krantz, and G. R. Jackson. 2007. A 845Drosophila model of mutant human parkin-induced toxicity demonstrates 846selective loss of dopaminergic neurons and dependence on cellular dopamine. 847J Neurosci 27:981-92. 848

58. Saydam, N., O. Georgiev, M. Y. Nakano, U. F. Greber, and W. Schaffner.8492001. Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 8501 is regulated by diverse stress signals. J Biol Chem 276:25487-95. 851

59. Schulz, J. B. 2008. Update on the pathogenesis of Parkinson's disease. J 852Neurol 255 Suppl 5:3-7. 853

60. Selvaraj, A., K. Balamurugan, H. Yepiskoposyan, H. Zhou, D. Egli, O. 854Georgiev, D. J. Thiele, and W. Schaffner. 2005. Metal-responsive 855transcription factor (MTF-1) handles both extremes, copper load and copper 856starvation, by activating different genes. Genes Dev 19:891-6. 857

61. Silar, P., L. Theodore, R. Mokdad, N. E. Erraiss, A. Cadic, and M. 858Wegnez. 1990. Metallothionein Mto gene of Drosophila melanogaster: 859structure and regulation. J Mol Biol 215:217-24. 860

62. Smirnova, I. V., D. C. Bittel, R. Ravindra, H. Jiang, and G. K. Andrews.8612000. Zinc and cadmium can promote rapid nuclear translocation of metal 862response element-binding transcription factor-1. J Biol Chem 275:9377-84.863

63. Southon, A., R. Burke, M. Norgate, P. Batterham, and J. Camakaris.8642004. Copper homoeostasis in Drosophila melanogaster S2 cells. Biochem J 865383:303-9. 866

64. Stuart, G. W., P. F. Searle, H. Y. Chen, R. L. Brinster, and R. D. 867Palmiter. 1984. A 12-base-pair DNA motif that is repeated several times in 868metallothionein gene promoters confers metal regulation to a heterologous 869gene. Proc Natl Acad Sci U S A 81:7318-22. 870

65. Tumer, Z., and N. Horn. 1998. Menkes disease: underlying genetic defect 871and new diagnostic possibilities. J Inherit Metab Dis 21:604-12.872

66. Valko, M., H. Morris, and M. T. Cronin. 2005. Metals, toxicity and 873oxidative stress. Curr Med Chem 12:1161-208. 874

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

32

67. Vandesompele, J., K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De 875Paepe, and F. Speleman. 2002. Accurate normalization of real-time 876quantitative RT-PCR data by geometric averaging of multiple internal control 877genes. Genome Biol 3:RESEARCH0034. 878

68. Wang, Y., I. Lorenzi, O. Georgiev, and W. Schaffner. 2004. Metal-879responsive transcription factor-1 (MTF-1) selects different types of metal 880response elements at low vs. high zinc concentration. Biol Chem 385:623-32. 881

69. Wang, Y., U. Wimmer, P. Lichtlen, D. Inderbitzin, B. Stieger, P. J. Meier, 882L. Hunziker, T. Stallmach, R. Forrer, T. Rulicke, O. Georgiev, and W. 883Schaffner. 2004. Metal-responsive transcription factor-1 (MTF-1) is essential 884for embryonic liver development and heavy metal detoxification in the adult 885liver. Faseb J 18:1071-9. 886

70. West, A. B., and N. T. Maidment. 2004. Genetics of parkin-linked disease. 887Hum Genet 114:327-36. 888

71. West, A. K., R. Stallings, C. E. Hildebrand, R. Chiu, M. Karin, and R. I. 889Richards. 1990. Human metallothionein genes: structure of the functional 890locus at 16q13. Genomics 8:513-8. 891

72. Whitworth, A. J., D. A. Theodore, J. C. Greene, H. Benes, P. D. Wes, and 892L. J. Pallanck. 2005. Increased glutathione S-transferase activity rescues 893dopaminergic neuron loss in a Drosophila model of Parkinson's disease. Proc 894Natl Acad Sci U S A 102:8024-9. 895

73. Winklhofer, K. F., and C. Haass. 2010. Mitochondrial dysfunction in 896Parkinson's disease. Biochim Biophys Acta 1802:29-44. 897

74. Yepiskoposyan, H., D. Egli, T. Fergestad, A. Selvaraj, C. Treiber, G. 898Multhaup, O. Georgiev, and W. Schaffner. 2006. Transcriptome response to 899heavy metal stress in Drosophila reveals a new zinc transporter that confers 900resistance to zinc. Nucleic Acids Res 34:4866-77. 901

75. Zhang, B., D. Egli, O. Georgiev, and W. Schaffner. 2001. The Drosophila 902homolog of mammalian zinc finger factor MTF-1 activates transcription in 903response to heavy metals. Mol Cell Biol 21:4505-14. 904

76. Zhang, B., O. Georgiev, M. Hagmann, C. Gunes, M. Cramer, P. Faller, 905M. Vasak, and W. Schaffner. 2003. Activity of metal-responsive 906transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by 907metallothionein. Mol Cell Biol 23:8471-85. 908

909

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

tub-MTF-1 +

CyO TM3,y+X

upon selfing

tub-MTF-1 park25, MTF-1-

CyO TM3,y+

;

Tub-MTF-1 park25, MTF-1-

CyO TM3, y+;

tub-MTF-1 park25, MTF-1-

tub-MTF-1 park25, MTF-1-; ;

tub-MTF-1 park25, MTF-1-

CyO park25, MTF-1-;tub-MTF-1 TM3

CyO TM3;

tub-MTF-1 TM3

tub-MTF-1 TM3;

;

Sp park25, MTF-1--

CyO TM6B;

Figure 1

Scheme of genetic cross to obtain strong MTF-1 expression on double mutant background. An MTF-1 cDNAtransgene driven by the tubulin promoter was combined with homozygous mutations of both MTF-1 (MTF-1-)(17) and parkin (park25) (24); progeny in red. The Sp marker on the second chromosome gives a uniform-length side-bristle

phenotype and the CyO second chromosome balancer results in curly wings. TM3 and TM6B are third chromosome balancers

which display a serrated wing phenotype and a dense side bristle phenotype, respectively. Balancers are lethal in homozygous

form; progeny marked with a cross indicate lethality of that particular genotype. Chromosome 1 is wild type.

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

A

B

Figure 2

Figure 2. Enhanced lifespan of parkin mutants

expressing an MTF-1 transgene. (A) A cDNA

transgene of Drosophila-MTF-1 driven by the

ubiquitously active tubulin enhancer/promoter

prolongs the lifespan of parkin mutants (park25/25)

up to 41 days. For survival of park25/25 vs.

tub-MTF-1, park25/25 flies, p<0.001. (B) Strong

expression of a UAS-MTF-1 transgene driven by

actin-Gal4 in a parkin mutant background also

extends lifespan, as tested in multiple sets of

Act-Gal4; UAS-MTF-1, park25/25 flies. In both (A)

and (B), heterozygous park25/+ flies with MTF-1

overexpression had a similar lifespan as park25/+

controls (data not shown).

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 3 legend on next page

A A’’A’

B B’’’B’’B’

C C’’C’

p

p

p

p

p

pp

pp

p

a

a

a

a

a

a

a

a

a

a

a

w; tub-MTF-1, park 25/+; +

w; tub-MTF-1, park 25/25; +

w;+; park 25/25

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 3. Elevated MTF-1 expression restores ovary structures and restores fertility of parkin mutant females. (A-

A’’) Normal ovariole structures of fertile control female flies (park25/+). (B-B’’’) Infertile female parkin mutants (park25/25)

have a distorted ovary structure with very few mature eggs. (C-C’’) Upon expression of a tubulin-driven MTF-1 transgene

ovary structures are normalised, resulting in normal fertility. a, anterior with germarium; p, posterior with vitellarium;

broken arrows indicate mature eggs; full arrows developing stages of eggs. Phalloidin (red) stains tubulin structures and

Toto (blue) stains the nuclei.

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

***

***

******

Figure 4. MTF-1 transgene expression restores the climbing ability of parkin mutants. Tubulin enhancer/promoter-

driven MTF-1 expression largely rescues the locomotion ability of park25/25 flies. In control flies, MTF-1 overexpression

does not further improve climbing ability. Data shown represents mean value ! standard deviation of each group tested

every other day up to 8 days. Asterisks show highly significant differences between parkin control (park25/+) and parkin

mutant (park25/25) flies on each day of the assay (p< 0.0001).

Figure 4

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

w; +; park25/+ w; +; park25/25w; Ac-Gal4; UAS-MTF-1,park25/25

2 μm

A

B E

D

C

H

G

F I

Figure 5

legend on next page

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 5. Strong MTF-1 expression improves muscle and mitochondrial morphology of parkin mutants. (A-C)

Transverse sections of indirect flight muscles (IFMs) show well preserved muscle in park25/+ heterozygous controls with

a regular myofibril arrangement (white arrows) and many electron-dense mitochondria (broken red arrows). (D-F) park25/25

adult IFMs show an irregular myofibrillar arrangement with diffuse Z-lines and M-bands and numerous vacuoles.

Mitochondria are swollen with fragmented cristae (red arrows). (G-I) Myofibril and mitochondrial integrity of parkin

mutants is restored by MTF-1 overexpression. Mitochondria are more dense (broken white arrows) and muscle structure

is more regular (broken green arrows), although occasional vacuoles are observed (green arrows). The scale bar is for top

and middle panels while the bottom panel is at a higher magnification, shown for clarity.

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

C DBA

Metal response

Element (MRE) EYFPMtnA promoter

-446 to +74

MtnA promoter/EYFP fusion

Figure 6. MTF-1 activity is upregulated in a parkin deficient background. Top: Transgenic MtnA/EYFP reporter gene (6).

Bottom: (A-B) MtnA-EYFP, park25/+ and (C-D) MtnA-EYFP, park25/25. Pictures of 1-2 day old adult flies were taken at 730

ms exposure with a Leica fluorescence microscope. ‘a’ and ‘p’ show anterior and posterior ends, respectively.

Figure 6

a

a

a

a

pp

p

p on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 7 (legend next page)

*** ***

******

******

*** ***

A C

B D

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 7. Increased MTF-1 and metallothionein (MtnB) transcript levels in parkin mutants that also express an

MTF-1 transgene. Real-time transcript-levels of (A) MTF-1, (B) MtnB, (C) parkin and (D) foi in park25/+ and park25/25

and w; tub-MTF-1, park25/25 and Act-Gal4; UAS-MTF-1; park25/+ and Act-Gal4; UAS-MTF-1; park25/25 flies with MTF-1

knockout flies as an appropriate control. All flies were raised on normal food (NF). MTF-1 null flies show no MtnB and

parkin null flies show no parkin transcripts. (B) MTF-1 overexpression from the tubulin enhancer/promoter or via the

UAS-Act-Gal4 system induced a 234-fold and 270-fold increase in MtnB transcripts, respectively. *** indicates significant

difference between parkin mutant adult flies and parkin mutants with elevated MTF-1 expression (p<0.0001).

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 8

B

Legend on next page

C

0

5 M

10 M

15 M

20 M

25 M

park 25/+

(NF)

tub-MTF-1;

park25/+

(NF)

tub-MTF-1;

park25/+

(BPS+BCS)

park 25/25

(NF)

tub-MTF-1;

park25/25

(NF)

tub-MTF-1;

park25/25

(BPS+BCS)

MTF-1-/-

(NF)

Flu

ore

scen

ce

inte

nsit

y(c

ps)

*****

***

***

***

park 25/25

park 25/+

A

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 8. MTF-1 reduces reactive oxygen species (ROS) levels in parkin mutants and restores metal homeostasis. (A)

park25/25 flies (NF) show high amounts of ROS. MTF-1 transgene expression, or treatment with chelators of redox-active

metals (BPS and BCS) reduce ROS levels. parkin heterozygous controls (park25/+) do not show significant differences in

ROS levels with or without MTF-1-overexpression or dietary supplementation with metal chelators. The MTF-1 knockout

shows the highest ROS level. ***, p<0.001 and **, p<0.01, chelator-supplemented compared to normal food for the same

genotype (black stars), or compared to the same treatment between genotypes (red stars). (B) Elevated MTF-1 expression in

a parkin mutant background restores normal basal levels of the essential trace metals copper, iron and zinc when flies are

raised on NF (normal food). (C) Differences in metal content between controls, park25/25 and tub-MTF-1, park25/25 flies are

largely leveled out by metal supplementation of the food.

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Figure 9

Figure 9. Strong expression of human MTF-1 or of Drosophila metallothionein prolongs lifespan of parkin mutants.

A human-MTF-1 transgene driven by actin-Gal4 extends the median lifespan of parkin mutant flies from 7 (red) to 19 days

(light blue). Direct overexpression of metallothionein MtnA by tubulin promoter also enhances the median lifespan of

parkin mutants up to 17 days (green). An even better extension up to a median of 21 days is observed with a tubulin-driven

trangene of Drosophila MTF-1 (dark blue). park25/+ (black) and park25/25 (red) serve as controls.

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

19.0841668Cross 3

19.025849209Total

19.4981979Cross 2

18.4761462Cross 1

Percentage of parkin

and MTF-1

homozygous progeny

Total number of

progeny

Parkin and MTF-1

homozygous progeny:

w; Sp/CyO; MTF-1-/--,park25/25

Parkin and MTF-1

heterozygous progeny:

w; Sp/CyO; MTF-1-,

park25/ TM3

w; Sp/CyO; MTF-1-, park25/ TM3 X w; Sp/CyO; MTF-1-, park25/ TM315 mM NAC

supplemented

food

13.926637229Cross 3

15.315112311280Total

16.0882141741Cross 2

14.636353310Cross 1

Percentage of parkin

and MTF-1

homozygous progeny

Total number of

progeny

Parkin and MTF-1

homozygous progeny:

w; +; tub-MTF-1, park25/25

Parkin and MTF-1

heterozygous progeny:

w; +; tub-MTF-1, park 25/+

w; +; tub-MTF-1, park 25/+ X w; +; tub-MTF-1, park 25/+normal food

Table 1A

Table 1B

Table 1: footnote on next page

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from

Table I. (A) Partial rescue of synthetic lethality of combined parkin and MTF-1 mutants by

N-acetylcysteine: MTF-1, parkin heterozygous parents were crossed inter se on food supplemented with 15mM NAC.

(B) Increased frequency of flies reaching adulthood among parkin mutants overexpressing MTF-1. park25/25

mutants were obtained from parkin heterozygous parents crossed inter se. In both (A) and (B), egg laying was allowed

for two days by equal numbers of parents and eclosing progeny flies were counted thereafter. Three independent crosses

were done.

on January 6, 2019 by guesthttp://m

cb.asm.org/

Dow

nloaded from