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GENE-36769; No. of pages: 8; 4C: 4, 5, 6

Contents lists available at ScienceDirect

Gene

j ourna l homepage: www.e lsev ie r.com/ locate /gene

Diminution of eIF4E activity suppresses parkin mutant phenotypes

Cristina Ottone a,b, Alessia Galasso c, Marica Gemei c, Viviana Pisa a,d, Silvia Gigliotti c, Federica Piccioni a,Franco Graziani c,⁎,1, Arturo Verrotti di Pianella a,d,1,2

a CEINGE-Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples, Italyb SEMM-European School of Molecular Medicine, Via Comunale Margherita 482, 80145 Naples, Italyc Institute of Genetics and Biophysics “A. Buzzati Traverso”, CNR, Via P. Castellino 111, 80131 Naples, Italyd Dipartimento di Biochimica e Biotecnologie Mediche, Via S. Pansini 5, 80131 Naples, Italy

Abbreviations: AR-JP, autosomal recessive-juvenileukaryotic translation initiation factor 4E; m7GpppN,poly(A) binding protein; PARK2, human parkin; 4E-BP,eukaryotic translation initiation factor G; GST, Glutatdalton(s); TOR, target of rapamycin; DA neurons,Parkinson's disease.⁎ Corresponding author. Tel.: +39 0816132359.

E-mail address: graziani@igb.cnr.it (F. Graziani).1 Equally contributed to this work as corresponding a2 A.V. di P. dedicates this work to his father.

0378-1119/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.gene.2010.09.003

Please cite this article as: Ottone, C., et al.,gene.2010.09.003

a b s t r a c t

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Article history:Accepted 3 September 2010Available online xxxx

Received by A.J. van Wijnen

Keywords:DrosophilaOogenesisParkinTranslation initiation

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Mutations in the human parkin (PARK2) gene cause autosomal recessive-juvenile Parkinson's disease (AR-JP).In Drosophila melanogaster, mutant parkin alleles display a broad range of phenotypic alterations, includingfemale infertility. Here we report that reducing the level of eukaryotic translation initiation factor 4E (eIF4E)activity specifically rescues the female sterile phenotypes associated with the parkinP23 mutant allele.Additional defects, including reduction of pupal viability and body size, are also entirely recovered in bothmale and female flies of the abovementioned genotype. We further show that a null eIF4E-binding protein (4E-BP) allele counteracts the in vivo effects produced, in a parkinP23 mutant background, by the reduction offunctional eIF4E copy number. Moreover, Parkin and eIF4E interact in vitro and co-localize at the posterior endof developing oocytes. Finally, we show that eIF4E is over-expressed in parkinP23 mutant ovaries as comparedto wild-types. Taken together, our data are consistent with the idea that Parkin and eIF4E act in a commonpathway, likely modulating cap-dependent translation initiation events.

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e Parkinson's disease; eIF4E,m7GTP cap structure; PABP,eIF4E-binding protein; eIF4G,ione-S-Transferase; kDa, kilo-dopaminergic neurons; PD,

uthors.

l rights reserved.

Diminution of eIF4E activity suppresses parki

© 2010 Elsevier B.V. All rights reserved.

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1. Introduction

Mutations in the evolutionary conserved human parkin (PARK2)gene cause autosomal recessive-juvenile Parkinson's disease (AR-JP)(Kitada et al., 1998; Giasson and Lee, 2003). Since Parkin acts as an E3ligase by interacting with E2 ubiquitin-carrier (Ubc) proteins(Shimura et al., 2000; Zhang et al., 2000; Staropoli et al., 2003), theloss of its activity is thought to lead to neurodegeneration because ofthe progressive accumulation of non-ubiquitinated, thus probablytoxic substrates.

A number of putative Parkin substrates have been proposed inmammals including Pael-R (Parkin-associated endothelin receptor-likereceptor) (Imai et al., 2001), synphilin (Chung et al., 2001), O-glycosylated α-synuclein (Shimura et al., 2001), Cyclin E (Staropoli etal., 2003),α/β-tubulins (Ren et al., 2003), RanBP2 (Umet al., 2006), andβ-amyloid (Rosen et al., 2009). These observations raise the possibility

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that Parkin might be involved in processes as diverse as cell signaling,synaptic functions, and cell cycle control.

Parkin-deficient mice do not appear to be robust animal models tostudy AR-JP (Perez and Palmiter, 2005). On the contrary, Drosophilastrains bearing null parkin alleles exhibit a complex set of defectswhich, depending on the mutant allele analyzed, range from partialpupal lethality to female and male sterility, reduced longevity,abnormal wing posture, defects in flying and climbing, reducedbodymass, cell size and number, increased sensitivity to chemical andenvironmental stress, degeneration and shrinkage of a subset ofdopaminergic neurons in the brain (Greene et al., 2003; Pesah et al.,2004; Cha et al., 2005; Whitworth et al., 2005). Even if differentresearch groups have reported diverse effects of parkin loss-of-function on the dopaminergic neurons, all results indicate thatmitochondrial defects are the earliest cellular manifestation of thetissue degeneration in the mutants, suggesting a role for Parkin inmitochondrial integrity (Riparbelli and Callaini, 2007) and DNA repair(Rothfuss et al., 2009). Indeed, loss of Drosophila PTEN-inducedputative kinase 1 (PINK1) leads to mitochondrial dysfunction that isrescued by over-expression of Parkin (Clark et al., 2006; Park et al.,2006; Yang et al., 2006). Thus, PINK1 and Parkin, the two genes mostcommonly associated with AR-JP, appear to function in a commonpathway.

Since Parkin is involved in ubiquitination pathways (Giasson andLee, 2001), it is reasonable to predict that loss of Parkin activity mightresult in the progressive accumulation, in different tissues (including

n mutant phenotypes, Gene (2010), doi:10.1016/j.

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Table 1 t1:1

Reducing eIF4E copy number rescues the parkinP23 pupal semi-lethality.t1:2t1:3Crosses Total

progeny% of parkP23

homozygousprogeny expected

% of parkP23

homozygousprogeny observed

t1:4parkinP23/TM3×parkinP23/TM3 301 33 4.4±1.5t1:5parkinP23, eIF4E07238/TM3×

parkinP23/TM31439 33 32.3±6.7

Pb0.001. t1:6

Table 2 t2:1

Rescue of parkinP23 female sterility by diminishing eIF4E copy number.t2:2t2:3Genotype No. of

depositedeggs

No. ofmothers(average)

Eggs/mother

No. oflarvae

% eclosion

t2:4CS 1293 10.7 129.4 977 75.50t2:5parkinP23/parkinP23 11 8.7 1.2 0 0t2:6parkinP23/parkinP23,

eIF4E07238407 9.5 42.8 165 40.50

2 C. Ottone et al. / Gene xxx (2010) xxx–xxx

dopaminergic neurons and, at least in Drosophila, germ-line cells), ofproteins that are actively translated, but cannot be properly degraded.Thus, the translational machinery could play a crucial role inameliorating/worsening of the parkin mutant phenotypes.

To investigate this hypothesis, we focused on cap-dependenttranslation initiation. The eukaryotic translation initiation factor 4E(eIF4E) binds directly the cap structure, present at the 5′ end of allmRNAs transcribed in the cell nucleus, and promotes translationinitiation (Piccioni et al., 2005). In Drosophila, eIF4E07238 loss-of-functionmutants arrest growth during larval development, while flieswith a point mutation in the major phosphorylation site of eIF4E(eIF4ESer251Ala) are viable, but display female sterility, smaller bodysize, delayed development, and eyes with ommatidia reduced in bothsize and number (Lachance et al., 2002). Given the similar phenotypiccharacteristics of few parkin alleles and eIF4ESer251Ala mutant alleles,we asked whether parkin and eIF4E genetically interact. Therefore, weintroduced on the same 3rd chromosome, by means of meioticrecombination, both parkinP23 and eIF4E07238 mutant alleles.

In this report, we show that homozygous parkinP23 female fliescarrying a single functional copy of the eIF4E gene are fully fertile withnormal-size ovaries when compared to parkinP23/parkinP23 females.Other phenotypes, such as partial pupal lethality and reduced bodysize, of the parkinP23 mutant flies are also fully rescued in both malesand females. Interestingly, the rescue is significantly counteracted in anull eIF4E-binding protein (4E-BP) genetic background. In addition,Parkin and eIF4E interact biochemically and co-localize within theposterior cytoplasm of developing oocytes. Finally, eIF4E is over-expressed in parkinP23 mutant ovaries. Our results suggest thatspecific modulation of cap-dependent translation at the initiationlevel affects the severity of several parkin defects.

2. Materials and methods

2.1. Drosophila strains and crosses

Flies were raised at 25 °C on standard sucrose/cornmeal/yeastfood.

parkinP21 and parkinP23 are described byPesahet al. (2004), eIF4E07238

is described by Lachance et al. (2002), and 4E-BPnull is described byTettweiler et al. (2005).

parkin stocks used for rescue experiments of parkinP23 mutant aredescribed in Pesah et al. (2004).

To obtain parkinP23, eIF4E07238 recombinant chromosomes, parkinP23/TM3 females were crossed to eIF4E07238/TM3males. parkinP23/eIF4E07238

virgin females were crossed in mass to TM3/+ males. After matingsingly the TM3 male offspring with females carrying double balancedthird chromosome, each progeny was examined for parkinP23,eIF4E07238

recombinants. Therefore, PCR analyses were performed using two setsof primers: for each set, one primer was complementary to the P-wingand the other to parkin or eIF4E gene sequences respectively. The stockscontaining the parkinP23,eIF4E07238 recombinant chromosomes andthose carrying only the parkinP23 mutation were collected.

Mass analysis was performed as follows: twenty individual malesand females were weighed on precision scale (range 0.01 mg to 50 g;Mettler Toledo AG245). In addition, groups of 20 or 40 flies of eachgenotype were weighed to confirm accuracy. Flies were reared underthe same conditions and age matched.

To calculate the pupal lethality (Table 1), parkinP23/TM3,Sb virginfemales were crossed to either parkinP23/TM3,Sb or parkinP23,eIF4E07238/TM3,Sbmales and the progeny scored for Sb or not Sbphenotypes, everyday for 10 days at 25 °C. The percentage of non Sb progeny wascalculated on total progeny. The standard error was also estimated andsignificance determined by one way ANOVA.

To assess fertility (Table 2), wild-type Canton-Smalesweremated toeitherparkinP23/parkinP23orparkinP23,eIF4E07238/parkinP23virgin females.The eggs, laid at 25 °C on fresh yeast-agar plates containing apple juice,

Please cite this article as: Ottone, C., et al., Diminution of eIF4E activitygene.2010.09.003

were counted every day for 12 days. The survival females were alsocounted every day and the number reportedwithin the table representsthe average. Every plate, after egg deposition,wasmaintained for 5 daysat 25 °C and carefully scored for emerging larvae.

2.2. GST pull-down assay and Western blots

The plasmid encoding GST–Parkin was generated by inserting aDraI–XhoI parkin cDNA fragment (encoding amino acids 6–482 andderived from pOT2-parkin) into pGEX-4T-2, digested with SmaI andXhoI.

Total protein extracts from adult ovary pairs were prepared aspreviously described (Kim-Ha et al., 1995).

Binding reactions were performed as previously described (Ver-rotti and Wharton, 2000), except that ovary extracts were incubatedwith 2 μg of either GST–Parkin or GST alone, bound to glutathioneagarose beads, for 2 h at 4 °C.

eIF4E was detected by Western blot using a rabbit anti-eIF4Eantibody (Lachance et al., 2002).

2.3. DNA staining and immunocytochemistry

Whole ovaries were hand-dissected and fixed as previouslydescribed (Gigliotti et al., 1998). Protein localization was visualizedby using rabbit anti-Parkin (CIM Antibody Core, Arizona StateUniversity) and rabbit anti-eIF4E antisera. Donkey anti-rabbit Alexa488 IgGs (Molecular Probes) were used as secondary antibodies.Images were generated with either a Leica Laser Scan or a ZeissLSM510 Meta confocal microscope.

After staining with DAPI to visualize DNA, ovaries were analyzedby conventional epifluorescence microscopy. Nomarski optics wereused to compare ovary pair size of different genotypes.

3. Results

3.1. parkin ovarian phenotypes

Drosophila oogenesis (Spradling, 1993) starts in the germarium,which contains two types of stem cells generating germ-line andsomatic follicle cells. The germ-line stemcells divide asymmetrically togenerate a stem cell and a cystoblast. The latter divides four times toproduce a 16-cell syncytial cyst andoocyte specification initiates in oneof these cells. The other 15 germ-line derived cells differentiate intotrophic nurse cells. An egg chamber is formed when about 80 somatic

suppresses parkin mutant phenotypes, Gene (2010), doi:10.1016/j.

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3C. Ottone et al. / Gene xxx (2010) xxx–xxx

cells migrate to surround a single germ-line cyst. The remainingsomatic cells produce a continuous epithelial sheet of follicle cellsaround the germ-line cyst. The egg chamber subsequently buds fromthe germarium and enters in the vitellarium, where it progressesthrough 14 developmental stages. During stage 9, most follicle cellsmigrate posteriorly to cover the oocyte surface in a columnarepithelium. At the beginning of stage 10B, subgroups of these cellschange their shape and migrate centripetally, moving inward at theboundary between nurse cells and the oocyte. During stage 11, theoocyte rapidly enlarges with the transfer of the nurse cell contents, aprocess called nurse cell dumping.

In Drosophila, few parkin mutant alleles, known to be null, havebeen reported to be female infertile (Pesah et al., 2004). Their ovarianphenotypes have never been described, however the female sterilityis fully rescued by a genomic fragment containing the parkin gene(Pesah et al., 2004). We, therefore, performed a detailed morpholog-ical analysis of the whole oogenesis process in parkinmutant flies. Ourpreliminary analyses have been focused on two different mutantalleles, parkinP21 and parkinP23, generated by P-element insertion indifferent regions of the parkin gene (Pesah et al., 2004). The twoalleles affect oogenesis to a different extent, as expected from thenature of their molecular lesions. parkinP21 is able to sustain egg-chamber maturation until the production of a mature oocyte, even ifthe laid eggs are unable to develop further. In contrast, parkinP23 ismore severe and induces an early developmental block, mostlyresulting in the generation of pre-vitellogenic stages (prior to stage 8)and only rare mature egg chambers.

In parkinP21, we observed a prominent defect in the rapid phase ofcytoplasm transfer from the nurse cells to the oocyte. Due to the blockof this process, the nurse cells fail to regress and persist until thecompletion of oogenesis, when eggshell morphogenesis culminates inthe synthesis of the dorsal appendages. Therefore, at the end ofoogenesis the mature egg acquires a cup-like shape and displays astrong reduction in size (Fig. 1, observe the same egg chamber inpanels C and D).

Fig. 1. Ovarian parkin phenotypes. A and B: DAPI-staining of wild-type egg chambers. A: stagmutant female flies; D: dark field of the same egg chamber displayed in C (note the cup-like shstaining of fused egg chambers,with amultiple number of germ cells, derived from parkinP23muon scale.

Please cite this article as: Ottone, C., et al., Diminution of eIF4E activitygene.2010.09.003

In parkinP23, large, abnormal egg chambers, containing more than16 germ-line cells, are produced (Fig. 1, panels E–F). Most of theselarge follicles appear to be formed by multiple 16-germ cell cysts,enveloped by a single follicle cell epithelium, because: a) they containtwo or more groups of 15 nurse cells, which are temporallycoordinated in their development; b) the nurse cells to oocyte ratioremains 15:1; c) normal cystocyte interconnections are present,based on phalloidin staining that visualizes the actin cytoskeleton(data not shown). In egg chambers containing a number of germ cellsmultiple of 16, one oocyte is usually located at the posterior while theother oocytes are located either anteriorly or medially. Occasionally,abnormal egg chambers that contain less than 16 germ-line cells arealso produced. We fully rescued all these phenotypes by using agenomic fragment bearing the wild-type parkin gene and 10–12 kbflanking it on either side, thus showing specificity of the above-mentioned ovarian phenotypes (data not shown). We would like tomention that the pupal semi-lethality and the reduced body size,displayed by homozygous parkinP23, are also fully rescued.

Taken together, these phenotypes suggest that parkin function isrequired for normal follicle cell proliferation and/or migration, which,in turn, is necessary for normal oocyte maturation.

3.2. Construction of chromosomes carrying mutations in both parkin andeIF4E

Even if a total of seven genes, encoding eight eukaryotic translationinitiation factor 4E (eIF4E) mRNA isoforms, have been identified(Hernandez et al., 2005), the two major and most studied mRNAisoforms, expressed during oogenesis and embryonic development upto larval stages, are transcribed from the eIF4E gene. Thus, in order topinpoint the molecular pathway(s) directing Parkin function, wedecided to analyze amelioration/worsening of parkin phenotypes byreducing in parkin flies the copy number of eIF4E. Since parkin andeIF4E genes reside on the same 3rd chromosome arm (bands 78C2 and

es 1 to 5; B: stage 10B; C: DAPI-staining of stage 13 egg chamber derived from parkinP21

ape of themature oocyte and the presence of the two dorsal appendages); E and F: DAPI-tant femaleflies. Note that anterior is on the left and posterior on the right. Images are not

suppresses parkin mutant phenotypes, Gene (2010), doi:10.1016/j.

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4 C. Ottone et al. / Gene xxx (2010) xxx–xxx

67B4 respectively), we produced, by means of meiotic recombination,chromosomes carrying mutations in both genes.

Among several existing mutant alleles of both genes, we focused onparkinP23 and eIF4E07238. parkinP23, generated by P-element insertion inthe coding region of the parkin gene (Pesah et al., 2004), is characterizedby an early block of oogenesis as described in the previous section;eIF4E07238, generated by P-element insertionwithin the largefirst intronof the eIF4E gene (Lachance et al., 2002), is homozygous lethal. Since thetwo mutants did not carry visible markers, the choice of these two Palleles facilitated the molecular screen of recombinant chromosomescarrying both mutations (see Materials and methods).

Six parkinP23,eIF4E07238 recombinants were obtained out of 50tested individuals and three independent recombinants were indi-vidually examined for amelioration/worsening of several parkinphenotypes. In these analyses, the recombinant individuals werealways compared to non recombinant parkinP23 siblings, in order toavoid any possible interference due to different genetic backgrounds.

3.3. Reduction of eIF4E gene activity rescues parkin pupal lethality anddiminished body size

parkin mutant individuals are homozygous viable, but eclose laterthan heterozygous controls. In addition, lack of Parkin protein leads toextensive pupal lethality (Pesah et al., 2004), which appears to resultfrom muscle dysfunction (Whitworth et al., 2005), even though littleor no lethality has been observed prior to pupation. We therefore firstexamined our recombinant individuals for the semi-lethalityphenotype.

The number of adult parkinP23 homozygous individuals, emergingfrom standard crosses between parkinP23/TM3 males and females, isvery low and shows a robust deviation from the expected mendelianratio (4,4% instead of 33%; Table 1), in agreement with previousobservations (7.2% instead of 33%; (Pesah et al., 2004)). In parallel, thenumber of parkinP23,eIF4E07238/parkinP23 flies emerging from crossesbetween parkinP23,eIF4E07238/TM3 virgin females and parkinP23/TM3males was determined and compared to the number of parkinP23,eIF4E07238/TM3 siblings. As summarized in Table 1, reducing the levelof eIF4E activity fully rescues the pupal semi-lethality associated withthe parkinP23 allele, thus demonstrating that parkin and eIF4E interactgenetically.

An additional consequence of parkin gene inactivation is a significantreduction of body size in both males and females when compared towild-type controls (Pesah et al., 2004).We therefore also compared themass of parkinP23,eIF4E07238/parkinP23 flies to the mass of homozygousparkinP23 flies of the same age and sex. parkinP23mutant animals displaya mass reduction of approximately 30% in both sexes. This defect is

Fig. 2. Recovery of reduced parkinP23 body size by lowering eIF4E copy number. (A) Photmeasurement of 20 individual females was performed (Pb0,001). Flies were reared under

Please cite this article as: Ottone, C., et al., Diminution of eIF4E activitygene.2010.09.003

almost completely rescued by lowering eIF4E activity (Fig. 2), furthersupporting the genetic interaction data reported above.

3.4. Reduction of eIF4E gene activity ameliorates ovarian parkinphenotypes

Since the reduction of eIF4E copy number, in a parkin homozygousgenetic background, results in rescue of both the pupal semi-lethalityand body size phenotypes, we tested whether lowering the level ofeIF4E rescues also the female sterility and/or the ovarian defectsassociated with the parkinP23 mutation.

We therefore mated parkinP23,eIF4E07238/parkinP23 and parkinP23/parkinP23 virgin females with wild-type Canton-S males and deter-mined the number of eggs laid per mother and the percentage of eggeclosion over a 12-days period. As summarized in Table 2, parkinhomozygous female flies bearing only one copy of functional eIF4Eshow an improvement of both parameters: the number of matureoocytes oviposited (1.2/mother in the case of parkinP23/parkinP23 vs.42.8/mother in the case of parkinP23,eIF4E07238/parkinP23 females), andthe rate of larval hatching (0% vs. 40.5%). These values, even thoughdistant from those obtained using wild-type Canton-S females (150eggs/mother and 75% of larval hatching), indicate that the reductionof eIF4E copy number substantially restores the fertility of parkinP23

females.In addition, we examined the morphology of parkinP23,eIF4E07238/

parkinP23 and parkinP23/parkinP23 egg chambers by Nomarski optics andepifluorescence microscopy observations. The introduction of a singleeIF4E07238 allele abrogates significantly the oogenesis defects inhomozygous parkinP23 mutant females. As shown in Fig. 3A, whileparkinP23 mutant ovaries contain only rare mature egg chambers, inparkinP23,eIF4E07238/parkinP23ovaries,most egg chambers appearnormalandwill develop into adult individuals. In agreementwith this result, thereduced size of parkinP23 ovaries is also rescued by diminishing the eIF4Ecopy number (Fig. 3B). Thus, reducing the level of eIF4E abolishes theoogenesis defects associated with the parkinP23 allele.

To demonstrate that the rescuing effects produced on the parkinP23

phenotypes by the reduction of eIF4E copy number are due to themodulation of translation, we tested the genetic consequences ofeliminating the activity of the eIF4E-binding protein (4E-BP), which isable to inhibit eIF4E. Flies completely lacking 4E-BP activity are viableunder normal growth conditions (Tettweiler et al., 2005). As shown inFig. 4, in parkinP23,eIF4E07238/parkinP23 individuals carrying 4E-BP nullalleles (4E-BPnull/4E-BPnull;parkinP23,eIF4E07238/parkinP23) the pupalsemi-lethality, linked to the parkinP23 chromosome, reappears asexpected. In addition surviving flies display a broad range of body size(data not shown),which is also indicative of restoration of the parkinP23/

omicrographs of 3 days-old females are all at the same magnification. (B) Body massthe same conditions and age matched.

suppresses parkin mutant phenotypes, Gene (2010), doi:10.1016/j.

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Fig. 3. Rescue of parkinP23 ovarian phenotypes. (A) DAPI-staining of ovarioles of the indicated genotypes. Arrows point to multi-nucleated cyst follicles. (B) Ovaries of the indicatedgenotypes dissected 3–5 days after eclosion. All photomicrographs are at the same magnification.

5C. Ottone et al. / Gene xxx (2010) xxx–xxx

parkinP23phenotype.Only theovarianphenotype, linked to theparkinP23

chromosome, does not reappear in the survival females, lacking the 4E-BP activity.

Taken together, our results indicate that the amelioration of theparkin phenotypes, observed in parkinP23,eIF4E07238/parkinP23 indivi-duals, is eIF4E-dependent and directly linked to a modification oftranslation initiation efficiency.

3.5. Parkin interacts with eIF4E

To pinpoint the in vivo localization of Parkin protein during egg-chamber development, we carried out immunostaining experimentson fixed wild-type ovaries using a polyclonal anti-Parkin antibody(Fig. 5). In the germarium, Parkin is specifically detectedwithin region2b, where follicle cell precursor proliferation takes place (Fig. 5, panelA). In early egg chambers, Parkin accumulates in the oocyte cytoplasmand continues to be selectively enriched in this cell until approx-imately stages 9–10. After these stages the staining is no longer

Fig. 4. Rescue of pupal semi-lethality in parkinP23, eIF4E07238/parkinP23 flies is counteractedby lack of 4E-BP activity. Percentage of non Sbprogenyderived from the following crosses:(A) parkinP23/TM3,Sb×parkinP23/TM3,Sb (901); (B) 4E-BPnull/4E-BPnull;parkinP23/TM3,Sb×4E-BPnull/4E-BPnull;parkinP23/TM3,Sb (1490); (C) parkinP23/TM3,Sb×parkinP23,eIF4E07238/TM3,Sb (1439); (D) 4E-BPnull/4E-BPnull;parkinP23/TM3,Sb×4E-BPnull/4E-BPnull;parkinP23,eIF4E07238/TM3,Sb (1947). Numbers of total progeny are in parentheses. Signi-ficance was determined by one way ANOVA.

Please cite this article as: Ottone, C., et al., Diminution of eIF4E activitygene.2010.09.003

observed. As depicted in Fig. 5 (panels B–D), the Parkin protein ispreferentially located at the posterior pole of wild-type developingoocytes. Moreover, it is specifically expressed in the anteriormargin ofthe oocyte (Fig. 5, panel D).

The Parkin protein is therefore mainly confined within thecytoplasm of growing oocytes, a pattern of expression that closelyresembles the previously reported eIF4E protein distribution (Zappa-vigna et al., 2004). In order to visualize both Parkin and eIF4E withinwild-type ovarioles, we performed double immunofluorescent anti-body stainings. Parkin and eIF4E co-localize to the posteriorcytoplasm of developing oocytes throughout oogenesis and up tostages 9–10. Fig. 6 (panels A–C) shows co-localization in a stage 5eggchamber, as a representative example. Thus, the spatiotemporaldistribution of the two proteins, at least within ovaries, is consistentwith the genetic interaction we observed.

Fig. 5. Immuno-localization by confocal microscopy of the Parkin protein in wild-typeegg chambers. A: Germarium (arrow indicates Parkin expression); B–D: Parkinaccumulates specifically in the developing oocyte until stage 9–10; B: stages 3 and 4;C: stage 6/7; D: stage 10 (arrowhead indicates the posterior pole of the oocyte). Notethat anterior is on the left and posterior on the right. Images B, C, and D are on scale.

suppresses parkin mutant phenotypes, Gene (2010), doi:10.1016/j.

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Fig. 6. Immuno-localizationof ParkinandeIF4Eduringoogenesis. PanelsA,B, andC:Parkinand eIF4E co-localize during wild-type egg-chamber development. Panel D: eIF4Elocalization is maintained in parkinP23 ovaries (DAPI-staining demonstrates the integrityof the nuclei). Stage 5 egg chambers are shown as representative examples.

6 C. Ottone et al. / Gene xxx (2010) xxx–xxx

We then analyzed the distribution pattern of eIF4E in parkinP23

homozygous mutant egg chambers. eIF4E localization inside theoocyte remains basically unaltered (Fig. 6, panel D), whereas its levelsappear increased.We therefore compared the total amount of eIF4E inwild-type and parkinP23 homozygous ovaries byWestern blot. Ovariesderived from either mature or newly eclosed wild-type adult females(the latter consist, as the majority of parkinP23 ovaries, of eggchambers not older than stage 8) contain lower amounts of eIF4E ascompared to ovaries from parkinP23 flies (Fig. 7A). These resultsdemonstrate that Parkin is not necessary for the correct localization ofeIF4E within growing oocytes, but indicate that the absence of Parkinpromotes the augmentation of this translation initiation factor in thesame in vivo compartment.

Fig. 7. Parkin interacts with eIF4E in vivo and in vitro. (A) eIF4E is over-expressed in parkinP

type (lane 2), andmature parkinP23/parkinP23 (lane 3) females were analyzed byWestern blotusing an anti-α-tubulin antibody. (B) Parkin associates with eIF4E in a GST pull-down assretained proteins were eluted, and analyzed by SDS-PAGE. The arrow indicates the eIF4E p

Please cite this article as: Ottone, C., et al., Diminution of eIF4E activitygene.2010.09.003

Since the two proteins co-localize in vivo, at least in growingoocytes, we verified whether or not Parkin and eIF4E interactbiochemically. To this aim, we prepared a GST fusion protein bearingamino acids 6–482 of Parkin and incubated it with wild-type ovaryextracts. As shown in Fig. 7B, approximately 40% of eIF4E contained inthe extracts is retained by GST–Parkin under the used experimentalconditions (see Materials and methods), whereas an insignificantamount of eIF4E is retained in a control reaction using GST alone.These results indicate that Parkin and eIF4E are components of thesame multi-protein complex.

4. Discussion

In this report, we show that parkin and eukaryotic translationinitiation factor 4E (eIF4E) interact genetically and that the effects ofthis interaction are counteracted by mutations in the eIF4E-bindingprotein (4E-BP) gene. Moreover, Parkin and eIF4E are partners in acommon complex and co-localize in vivo. Our results furtherdemonstrate that eIF4E is over-expressed in parkinP23 homozygousmutants. Taken together, our results suggest that eIF4E-dependenttranslation contributes to the severity of several parkin mutantphenotypes in Drosophila.

4.1. Modulation of global translation initiation and autosomalrecessive-juvenile Parkinson's disease

All eukaryotic mRNAs possess a cap or m7GpppN (where N is anynucleotide andm amethyl group) structure and a poly(A) tail at the 5′and 3′ ends respectively. Cap-bound eIF4E physically interacts withthe scaffolding protein eIF4G, which in turn interacts with poly(A)-binding proteins (PABP) bound to the polyadenylated tail (Piccioni etal., 2005). The consequent circularization of the mRNA guaranteestranslation efficiency and recruitment of translation initiation factors,including the 40S ribosomal subunit that starts scanning the mRNA insearch of an AUG in the proper sequence context (Kozak, 1989).

Modulation of global cap-dependent translation can be accom-plished at the initiation level by a family of translational repressors,known as eIF4E-binding proteins (4E-BPs), (Gingras et al., 2001),

23 ovaries. Ovary extracts derived from mature wild-type (lane 1), newly eclosed wild-using a rabbit anti-eIF4E antibody. The amount of total protein extracts was normalizeday. GST–Parkin and GST alone were incubated with mature wild-type ovary extracts;rotein (approximately 37 kDa) identified with an anti-eIF4E antibody.

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7C. Ottone et al. / Gene xxx (2010) xxx–xxx

which compete with eIF4G for eIF4E-binding. Hyperphosphorylationof the 4E-BPs by the TOR (target of rapamycin) kinase abolishesbinding to eIF4E, thus stimulating cap-dependent translation initia-tion. Rapamycin, a known potent immunosuppressant and inhibitorof protein synthesis in mammals (Gingras et al., 2001), negativelyregulates TOR.

In Drosophila, the keymolecules modulating global cap-dependenttranslation are involved in oxidative stress, regulation of growth(Lachance et al., 2002) and life span (Kapahi et al., 2004; Tettweiler etal., 2005). In particular, augmented 4E-BP-binding to eIF4E, resultingin lower amount of eIF4E available for translation, promotesresistance to oxidative stress and increased life span (Tettweiler etal., 2005). Similarly, TOR modulates life span in C. elegans (Jia et al.,2004) and the inhibition of TOR signaling by rapamycin can increaselife span in mammals, thus delaying death from cancer, retardingmechanisms of aging, and inhibiting the pathogenesis of late lifeillnesses (Harrison et al., 2009).

Translational control plays a key role during early development inmetazoa (Piccioni et al., 2005) and increasing lines of evidence showthat it is crucial to guarantee physiological functions and efficacy inneurons, thus suggesting its involvement, when altered, in neurode-generative diseases including Alzheimer's and Parkinson's (Chang andChou, 2006). In fact, each neuron might have the ability to modulatethe overall level of translation and/or translate a set of specific mRNAs(Sutton and Schuman, 2005).

Our work establishes a link between the eukaryotic translationalmachinery and one of the molecules known to be involved in thepathogenesis of Parkinson's disease (PD). In fact, diminishing eIF4E copynumber suppresses the oogenesis defects displayed by homozygousparkinP23 mutant females. As far as the germ-line is concerned, it isworth noting that the sterility of parkinP23/parkinP23 adult males is notrescuedby reducing eIF4E activity (data not shown). This result suggeststhat spermatogenesis is particularly sensitive to the accumulation oftoxic products, thus renderingmodulation of translation ineffective. Onthe contrary, the viability deficits displayed by homozygous parkinP23

individuals are rescued in both males and females (Table 1).It is important to point out that our experiments demonstrate a

bona fide genetic interaction between parkin and eIF4E. Indeed, we canexclude the influence of the fly genetic background, in particular thepresence of a parkin suppressor, because: 1) All six parkinP23,eIF4E07238

recombinant chromosomes, we obtained and analyzed, show identi-cal phenotypes in combination with three distinct parkinP23 nonrecombinant chromosomes (females are viable, fertile and normal-sized, and no pupal semi-lethality is observed); 2) All parkinP23 nonrecombinant chromosomes maintain all the parkin phenotypes whenin homozygous condition.

The parkin–eIF4E genetic interaction described above suggeststhat amelioration of the parkin phenotypes is related to modulationof eIF4E-dependent translation initiation. In line with this hypoth-esis, we demonstrated that the introduction of two copies of a 4E-BP null allele is able to counteract the effects of the eIF4E07238 allelein a parkinP23 homozygous background (4E-BPnull/4E-BPnull;parkinP23,eIF4E07238/parkinP23).

In light of our results, it remains to be ascertained whether or not areduction of eIF4E copy number may rescue the degeneration ofdopaminergic neurons in parkin mutant brains. Unfortunately, theparkin mutant allele utilized in this work does not reveal any loss ofdopaminergic neurons (Pesah et al., 2004), thus we were unable toverify our hypothesis in the fly brain. In addition, previous findingsfavor the idea that, in flies, parkin is not necessary for the survival ofDA neurons at least for three weeks after eclosion (Greene et al.,2003). Only later data showed that a subset of dopaminergic neuronsdegenerate or shrink in the brain of specific Drosophila parkin mutantalleles (Cha et al., 2005; Whitworth et al., 2005).

The loss of Parkin activity is thought to lead to neurodegenerationbecause of the progressive accumulation of non-ubiquitinated, thus

Please cite this article as: Ottone, C., et al., Diminution of eIF4E activitygene.2010.09.003

probably toxic substrates (Giasson and Lee, 2003). Therefore, the cause,development, and effects of autosomal recessive-juvenile Parkinson'sdisease (AR-JP) appear to resemble those of the autosomal-dominantform due to mutations in human α-synuclein (Schapira, 2009), even ifinclusions similar to Lewy bodies have never been observed neither inhuman nor in Drosophila parkin mutants.

To explain the remarkable amelioration of the parkin phenotypes,observed after reduction of eIF4E copy number, we can envision ascenario where diminished amount of functional eIF4E, available fortranslation, leads to lower accumulation of toxic substrates. However,the biochemical results presented in this work delineate a morecomplex picture implicating an intimate link between eIF4E andParkin proteins. Actually, they are components of a common proteincomplex and co-localize in vivo, at least during egg-chamberdevelopment, where eIF4E displays a distinctive subcellular localiza-tion at the posterior pole of growing oocytes. These data raise thepossibility that eIF4E itself is a Parkin substrate.

One hypothesis is that Parkin ubiquitinates eIF4E targeting it forsubsequent proteasome-dependent degradation. The presence ofaugmented eIF4E levels in parkin mutant ovaries supports our idea(Fig. 5B). Interestingly, Murata and Shimotohno (2006) suggestedthat mammalian eIF4E levels are down-regulated by ubiquitinationunder stress conditions. Moreover, Imai et al. (2008) have recentlyproposed that increased eIF4E-mediated protein translation, throughphosphorylation of 4E-BP by LRRK2 (leucine-rich repeat kinase 2),attenuates resistance to oxidative stress and survival of dopaminergicneurons in Drosophila. Indeed, mutations in the LRRK2 gene cause anautosomal-dominant form of familial PD late in life (Paisan-Ruiz et al.,2004; Zimprich et al., 2004). Furthermore, it has been recentlydemonstrated that over-expression of the translation inhibitor 4E-BPcan suppress all parkinmutant phenotypes, including degeneration ofdopaminergic neurons (Tain et al., 2009). It is therefore possible thatincreased sensitivity to stress, either chemical or environmental,observed in PD patients, is due, at least in part, to inefficientubiquitination and degradation of eIF4E. Since yeast two hybridassays failed to demonstrate direct binding between Parkin and eIF4E(data not shown), it is possible that Parkin acts on eIF4E in the contextof a multi-protein E3 complex. Indeed, selected RING finger E3 ligasesinteract directly with and ubiquitinate their substrates, while othersrequire additional factors to properly exert this function (Joazeiro andWeissman, 2000).

5. Conclusion

Our work shows that modulation of protein synthesis may have asignificant impact on Parkin-mediated phenotypes. As mentionedabove, translational initiation can be experimentally inhibited byrapamycin, via stimulation of 4E-BP activity. The data currentlyavailable for this compound and its analogs point to their ability toreduce lymphocyte proliferation and tumor cell growth, thussupporting the idea that they can be useful in cancer therapy (Gingraset al., 2001). Thus, in conjunction with recent findings (Tain et al.,2009), our current results suggest that these compounds might alsorepresent a valid therapeutic tool for the treatment of AR-JP andpossibly sporadic PD.

Acknowledgments

We would like to thank D. Andrenacci for critical reading of themanuscript; P. Bazzicalupo and U. di Porzio for helpful discussions.We also thank G. Mardon and P. Lasko for Drosophila stocks andantibodies; the DIM facility, in particular F. Formiggini, at Ceinge forimaging microscopy. We are also grateful to L.S. Pane who began thisproject and S. Andone for technical assistance.

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