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Article history:Received 27 September 2011Revised 16 December 2011Accepted 31 December 2011Available online 13 January 2012

Neurobiology of Disease 46 (2012) 137146

Contents lists available at SciVerse ScienceDirect

Neurobiology

j ourna l homepage: www.e lIntroduction

Parkinson's disease (PD) is characterized by the progressive, selec-tive loss of dopaminergic (DA) neurons of the substantia nigra parscompacta, formation of brillar cytoplasmic aggregates known asLewy bodies, dopamine depletion in the dorsolateral putamen anddebilitating motor impairments (Dauer and Przedborski, 2003). Theunknown etiology of this neurodegenerative disease and the lack ofeffective treatment highlight an urgent need for delineation of theunderlying pathological mechanisms.

While several genetic loci are associated with the rare familial form

pesticide exposure as a risk factor for the sporadic form of PD, whichconstitutes greater than 95% of all PD cases (Banerjee et al., 2009; Liouet al., 1997). The discovery that N-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) caused in humans an acute parkinsonism syndromebyinhibiting mitochondrial respiration at complex I of the electron trans-port chain (ETC) with its metabolite, 1-methyl-4-pyridinium (MPP+)provided an important link between mitochondrial dysfunction andPDpathogenesis (Tipton and Singer, 1993).MPTP induces neurotoxicityin animal brains by a mechanism of mitochondria-oxidative stress-energy failure (Fabre et al., 1999). Mice exposed to MPTP show reduc-tion in levels of bioenergetic metabolites ATP and NAD+ in the striatumof PD (Dawson et al., 2010), epidemiologica

Corresponding author. Fax: +1 860 486 5375.E-mail address: yih-woei.fridell@uconn.edu (Y.-W.C

1 Current address: Department of Entomology and NeGainesville, FL 32611, USA.

2 Those authors contributed equally to the work.Available online on ScienceDirect (www.scienced

0969-9961/$ see front matter 2012 Elsevier Inc. Alldoi:10.1016/j.nbd.2011.12.055 2012 Elsevier Inc. All rights reserved.Keywords:Human uncoupling protein 2 (hUCP2)Parkinson's Disease (PD)Dopaminergic (DA) neuronSpargelEnergy metabolismMitochondrial oxidative phosphorylationRotenoneParkinson's disease (PD), caused by selective loss of dopaminergic (DA) neurons in the substantia nigra parscompacta, is the most common movement disorder. While its etiology remains unknown, mitochondrial dys-function is recognized as one of the major cellular defects contributing to PD pathogenesis. Mitochondrialuncoupling protein 2 (UCP2) has been implicated in neuroprotection in several neuronal injury models.Here we show that hucp2 expression in Drosophila DA neurons under the control of the tyrosine hydroxylase(TH) promoter protects those ies against the mitochondrial toxin rotenone-induced DA neuron death, headdopamine depletion, impaired locomotor activity and energy deciency. Under normal conditions, hUCP2ies maintain an enhanced locomotor activity and have higher steady-state ATP levels suggesting improvedenergy homeostasis. We show that while no increased mitochondrial DNA content or volume fraction is mea-sured in hUCP2 ies, augmented mitochondrial complex I activity is detected. Those results suggest that it isincreased mitochondrial function but not mitochondrial biogenesis that appears responsible for higher ATPlevels in hUCP2 ies. Consistent with this notion, an up-regulation of Spargel, the Drosophila peroxisomeproliferator-activated receptor gamma coactivator 1 (PGC-1) homologue is detected in hUCP2 ies. Further-more, a Spargel target gene Tfam, the mitochondrial transcription factor A is up-regulated in hUCP2 ies.Taken together, our results demonstrate a neuroprotective effect of hUCP2 in DA neurons in a Drosophila spo-radic PD model. Moreover, as the TH promoter activity is present in both DA neurons and epidermis, our re-sults reveal that hucp2 expression in those tissues may act as a stress signal to trigger Spargel activationresulting in enhanced mitochondrial function and increased energy metabolism.a r t i c l e i n f o a b s t r a c tA neuroprotective role of the human uncoParkinson's Disease model

Raque Islam a,1,2, Lichuan Yang b,2, Megha Sah a, KaJenny Kwok a, Marie E. Cantino c, M. Flint Beal b, Yih-a Department of Allied Health Sciences, University of Connecticut, 358 Manseld Road, Storb Department of Neurology and Neuroscience, Weill Medical College of Cornell University,c Department of Physiology and Neurobiology, University of Connecticut, 75 N Eagleville Rol studies have suggested

. Fridell).matology, University of Florida,

irect.com).

rights reserved.pling protein 2 (hUCP2) in a Drosophila

ha Kannan a, Denise Anamani a, Chibi Vijayan a,oei C. Fridell a,T 06269, USAEast 68th Street, F-613, New York, NY 10065, USAStorrs, CT 06269, USA

of Disease

sev ie r .com/ locate /ynbd iand midbrain (Cosi and Marien, 1998). In C. elegans, ATP depletioncaused by the MPP+ induced mitochondrial dysfunction is a majortoxic effect resulting in DA neuron death and worm lethality (Wanget al., 2007). In addition to energy deciencies, oxidative stress is alsoassociated with mitochondrial complex I impairment and has beendetected in post-mortem studies and implicated in PD pathogenesis(Dexter et al., 1989; Mounsey and Teismann, 2010). For example, acommon pesticide rotenone has been shown to act on mitochondria

138 R. Islam et al. / Neurobiology of Disease 46 (2012) 137146complex I and result in ATPdepletion andoxidative damage in aneuronalcell culture system (Sherer et al., 2003). In a rodent PDmodel, rats chron-ically infused with rotenone subcutaneously were reported to developselective nigrostriatal DA degeneration, motor deciencies and oxidativedamage in the dopaminergic areas of the brain (Betarbet et al., 2000;Sherer et al., 2003). However, employing similar treatment conditions,additional studies have revealed variable effects in striatal TH immunore-activity and motor impairment raising concerns on the dopaminergicspecicity of rotenone treatment (Fleming et al., 2004; Hoglinger et al.,2003; Zhu et al., 2004). To address this variability, a more recent studyhas shown that intraperitoneal injection of rotenone appears to resultin reproducible PD associated motor defects, loss of TH-positive neuronsand-synuclein and poly-ubiquitin positive aggregates in DA neurons ofthe substantia nigra (Cannon et al., 2009). Similar to the rodent model,long-term exposure of Drosophila melanogaster (D. melanogaster) torotenone or paraquat has been shown to result in degeneration of DAneurons and loss of climbing ability (Chaudhuri et al., 2007; Coulomand Birman, 2004). While the impact on energy metabolism has notbeen determined under those treatments, these studies have validatedD. melanogaster as a relevant genetic model system in identifying molec-ular pathways contributing to sporadic PD pathogenesis.

Mitochondrial uncoupling proteins (UCPs) promote proton leak-age into the matrix thus disrupting the proton gradient generatedby the respiratory ETC, resulting in uncoupled substrate oxidationand ATP phosphorylation (Brand et al., 1999). While modulating en-ergy production, UCPs also decrease mitochondrial inner membranepotential, leading to lower proton motive force and decreased reac-tive oxygen species (ROS) production. The ndings that ucp2 expres-sion is induced following experimental procedures mimickingepilepsy (Sullivan et al., 2003), stroke (Mattiasson et al., 2003) andseizures (Diano et al., 2003) in mouse models suggest a neuroprotec-tive role of UCP2. Consistently, ucp2 expression in subpopulations ofneurons was found to be inversely proportional to activation of cas-pase 3, supporting the idea that UCP2 may counteract apoptotic celldeath in neurons (Mattiasson et al., 2003). To explore a role of UCP2in DA neuroprotection, transgenic approaches have shown that ubiq-uitous ucp2 overexpression protects animals from MPTP induced DAneuron loss and renders less oxidative damage in those neurons,whereas increased oxidative damage is detected in DA neuronsfrom ucp2 knock-out mice under the same treatment (Andrews etal., 2005). Similar approaches have revealed that increased mitochon-drial function mediated by UCP2 activity is necessary for the gut hor-mone ghrelin-mediated neuroprotection of DA neurons against MPTP(Andrews et al., 2009). In transgenic mice with catecholaminergicneuron-specic ucp2 overexpression, a partial neuroprotection isachieved following MPTP injection (Conti et al., 2005). However,while a reduction of oxidative markers was measured in UCP2 trans-genic animals, no changes in MPTP-induced oxidative damage weredetected between control and UCP2 transgenic animals (Conti et al.,2005). Thus, additional studies are necessary to understand themechanism underlying UCP2-mediated DA neuroprotection.

To address this question, we set out to investigate the neuroprotec-tive effect of hUCP2 against rotenone exposure in aDrosophila PDmodel(Coulom and Birman, 2004). We utilized our previously establishedUAS-hucp2 transgenic y line and generated transgenic TH>hUCP2ies harboring hucp2 expression under the control of the tyrosine hy-droxylase (TH) promoter. We show that rotenone-induced DA neuro-degeneration, head dopamine depletion and locomoter impairmentare attenuated in TH>hUCP2 ies. Interestingly, we demonstrate thatrotenone induced ATP deciency is less pronounced in those ies. Tofurther understand how energy metabolism may be modulated inTH>hUCP2 ies under normal conditions, we used molecular, cellularand behavioral assays to assess bioenergetic parameters includingmitochondrial content, mitochondrial ETC complex activity, steadystate ATP levels and climbing activity. Our results indicate that in-

creased mitochondrial function but not biogenesis appears responsiblefor the overall improved energy homeostasis in TH>hUCP2 ies. Con-sistent with this notion, real-time expression analysis of RNA isolatedfrom hUCP2 ies reveals an up-regulation of Spargel, theDrosophila ho-mologue of PGC-1 known as a major mitochondrial regulator and ROSsuppressor (St-Pierre et al., 2006). We further substantiate a functionalrole of Spargel in TH>hUCP2 ies by demonstrating an increased tran-script level of Tfam, the mitochondrial transcription factor A previouslyshown to be down-regulated in the fat body isolated from Spargel mu-tant larvae (Tiefenbock et al., 2010). In addition to its neuroprotectiveeffect in DA neurons against rotenone, hucp2 expression in DA neuronsand epidermis under the control of the TH promoter leads to broadphysiological effects in energy homeostasis, ROS metabolism and lifespan. A hypothesis proposing that both cell-autonomous and cell-non-autonomousmechanismsmay be responsible for the physiological out-comes in TH>hUCP2 ies will be discussed.

Results

hucp2 expression in DA neurons protects ies from rotenone-induced DAneuron death, head dopamine depletion, locomotor impairment and ATPdecit

To establish a neuroprotective role of hUCP2 in y DA neurons, werst determined whether exogenous hucp2 expression in DA neuronswould protect those neurons from rotenone induced DA neuron lossin a y PD model (Coulom and Birman, 2004). We employed thebipartite UAS-Gal4 expression system using our previously character-ized UAS-hucp2 transgenic line (Fridell et al., 2005, 2009) and the ty-rosine hydroxylase (TH)-Gal4 driver line where constitutive THpromoter activity is detected throughout development and adulthoodin both DA neurons and epidermis (Friggi-Grelin et al., 2003). Usingthose y lines, we obtained TH-Gal4/UAS-hucp2 ies as the experi-mental line (referred to as TH>hUCP2 ies hereafter). Since theUAS-hucp2 line was initially created in the w1118 genetic background,w1118 ies were used in parallel crosses to generate control TH-Gal4/w1118 ies for all studies (referred to as TH>w1118 ies hereafter)(Fridell et al., 2005, 2009). Using semi-quantitative RT-PCR analysis,we conrmed in our system that hucp2 transcript was only detectedin RNA isolated from heads of hUCP2 ies but not in heads isolatedfrom either UAS-hucp2 alone or control ies (Supplemental Fig. 1).

Chronic exposure to rotenone leads to degeneration of DA neuronsin the y (Coulom and Birman, 2004). To investigate whetherrotenone-induced DA neuron death may be attenuated in TH>hUCP2ies, we performed whole mount immunouorescence studies toquantify the number of DA neurons in adult brains following threedays of rotenone treatment. Distinct clusters of DA neurons in eachadult y brain hemisphere have been well documented (Friggi-Grelin et al., 2003; White et al., 2010). We also detected typical pat-terns of DA neurons using either an anti-TH antibody or by monitor-ing green uorescence staining in brains isolated from TH>GFP ies(data not shown). Following rotenone treatment, whole mountbrains from control and TH>hUCP2 ies were prepared and subjectedto immunostaining using an anti-TH antibody (Yang et al., 2006). Thenumber of TH+neurons detected in major clusters were thencounted and analyzed in a blind fashion (see Materials andMethods). As shown in Fig. 1A, a signicant DA neuron loss of 3051% was detected in four major clusters (PAL: 34%, PPM1/2: 31%,PPM3: 49% and PPL1: 51%) in control brains following rotenone treat-ment whereas an attenuated loss of those neurons was detected inTH>hUCP2 ies under the same treatment conditions (PAL: 26%,PPM1/2: 21%, PPM3: no detectable loss and PPL1: 4%). Thus, signi-cant neuroprotection against rotenone is achieved in TH>hUCP2ies in selective DA neuron clusters including PPM3 and PPL1. To un-derstand whether rotenone-induced DA neuron loss had a direct im-pact on brain dopamine content, we measured dopamine levels in

extracts from adult heads using HPLC (Faust et al., 2009). As shown

139R. Islam et al. / Neurobiology of Disease 46 (2012) 137146in Fig. 1B, an average of 25% reduction in dopamine content is detectedin the heads of rotenone-treated control ies whereas only a 9% reduc-tion is measured in TH>hUCP2 ies under the same treatment. Takentogether, we have shown that at the cell-autonomous level, hucp2expression in DA neurons protects those neurons from rotenone-induced cell death and depletion of head dopamine content.

Degeneration of DA neurons as the result of rotenone or paraquatexposure leads to motor deciencies (Chaudhuri et al., 2007; Coulomand Birman, 2004). Given an increased survival of selective DA neu-rons measured in TH>hUCP2 ies as compared to controls upon rote-none exposure, it was important to determine whether this DAneuroprotection resulted in less severe motor impairment in thoseies. Negative geotaxis assays have been successfully used to assessthe locomotor ability of ies (Coulom and Birman, 2004; Feany andBender, 2000). We adopted this procedure and demonstrated thatTH>hUCP2 ies retain a much better climbing ability by a range of30218% over a treatment period of 56 days as compared to geneti-cally matched control ies in the presence of rotenone (Figs. 1CD) orparaquat (Supplemental Fig. 2). Since rotenone is a mitochondrialcomplex I inhibitor resulting in ATP depletion in neuroblastomacells (Sherer et al., 2003), we next asked whether the better climbingability measured in TH>hUCP2 ies under rotenone treatment maybe associated with less affected energy production. To this end, we

Fig. 1. hucp2 expression in DA neurons protects ies from rotenone induced DA neuron degeneraDA degeneration is detected in selective clusters in brains isolated from TH>hUCP2 transmount adult brains were prepared and immuno-stained with an anti-TH antibody (Immuna blind fashion (see Materials and Methods). (B) Less dopamine depletion in TH>hUCP2mogenates of TH>hUCP2 and control ies reveals a 25% depletion of dopamine content in cmine content is detected in TH>hUCP2 ies under the same treatment for three days. N=1ability in control ies is attenuated in TH>hUCP2 ies. Ten-day-old ies were treated with2004; Feany and Bender, 2000). A better climbing ability by an average of 30218% was measConsistent results are measured in both male (C) and female (D) ies. The climbing abilitattained under treatment conditions (see Materials and Methods). N=12 vials with 20 i(Rot) treatment. Steady-state ATP levels were measured in TH>hUCP2 and control ies asreduction in ATP content was measured in control ies treated with rotenone whereas a 35%For all graphs, each value represents meanS.E.M. Statistical signicance between contr*pb0.05, **pb0.01. hUCP2: TH>hUCP2 ies; Control: TH>w1118 ies.measured steady-state ATP levels in TH>hUCP2 and control ies trea-tedwith rotenone as described (Park et al., 2006) and found that indeedrotenone-induced ATP depletion is less pronounced in TH>hUCP2 ies(Fig. 1E). Compared to their untreated counterparts, rotenone-treatedcontrol ies had a 63% reduction in ATP content whereas a 35% reduc-tion in total ATP content wasmeasured in rotenone-treated TH>hUCP2ies. Thus, in addition to the cell-autonomous, neuroprotective effect inDA neurons, TH>hUCP2 ies appear to maintain better energy homeo-stasis upon rotenone exposure. While the TH-Gal4 driver line has beenwidely used for studying genetic manipulations in y DA neurons, itspromoter activity is also present in the epidermis (Bayersdorfer et al.,2010; Friggi-Grelin et al., 2003). Thus, it is likely that expression ofhucp2 in the epidermis may also contribute to changes in ATP levelsmeasured in TH>hUCP2 ies.

TH>hUCP2 ies are more resistant to age-associated decline in climbingactivity and appear to maintain enhanced energy homeostasis

In light of our ndings that TH>hUCP2 ies retain a higher ATP con-tent than controls under rotenone treatment, it would be interesting tounderstand how energy metabolism may be modulated in TH>hUCP2ies under normal conditions. Interestingly, while performing betterin climbing activity assays under rotenone treatment, TH>hUCP2 ies

tion, head dopamine depletion, locomotor impairment and ATP deciencies. (A) Attenuatedgenic ies as compared to control brains. Following rotenone (Rot) treatment, wholeoStar Inc). N=1820 brains/condition. Scoring of the DA neurons was carried out inies as compared to controls following rotenone treatment. HPLC analysis of head ho-ontrol ies following rotenone (Rot) treatment whereas a 9% reduction in head dopa-6 samples (three heads/sample). (C and D) Rotenone induced impairment in climbing500 M rotenone and negative geotaxis assays were conducted (Coulom and Birman,ured in TH>hUCP2 ies than control ies throughout the course of rotenone treatment.y for control and TH>hUCP2 ies was determined while 50% or greater survival wases/vial. (E) A higher ATP content is maintained in TH>hUCP2 ies following rotenonedescribed in Materials and Methods. Compared to their untreated counterparts, a 63%reduction was detected in TH>hUCP2 ies under the same treatment. N=57 samples.ol and hUCP2 ies was determined using Student's t test (GraphPad Software, Inc.).

under control vehicle treatment also appeared to be more active thancontrol ies (data not shown). Therefore, we evaluated in more detailage-dependent climbing ability in both TH>hUCP2 and control ies.As shown in Figs. 2AB, while all ies exhibit age-associated decline intheir climbing ability, TH>hUCP2 ies maintained signicantly higherlevels of locomotor activity by an average of 2.9 fold in males and 2.4fold in females as compared to age and genetically matched controls.To correlate energy levels and the climbing ability measured inTH>hUCP2 and control ies, we quantied steady state ATP levels inthose ies as previously described (Park et al., 2006). As expected, anaverage of 44% increase in ATP levels was measured in TH>hUCP2 ascompared to control ies (Fig. 2C). In addition, wemeasured ATP levelsin UAS-hucp2 transgenic ies in the absence of the TH-Gal4 driver. Wedetected similar ATP levels between control and UAS-hucp2 ies indi-cating the physiological signicance of increased ATP levels inTH>hUCP2 ies (open bar in Fig. 2C).

Increased mitochondrial function in TH>hUCP2 ies

A higher ATP content measured in TH>hUCP2 ies suggests in-creased mitochondrial ATP production, which could be the result of in-creased mitochondrial biogenesis or enhanced mitochondrial function.To differentiate those two possibilities,we assessed parameters ofmito-chondrial bioenergetics. To evaluate mitochondrial biogenesis inTH>hUCP2 ies, we rst measured mitochondrial content by calculat-ing the ratio of mitochondrial DNA to nuclear DNA using quantitativereal-time PCR analysis. By quantifying the copy numbers of a

mitochondrial gene encoding cytochrome oxidase subunit I (COI) anda nuclear gene GAPDH, we detected no discernible changes in COI/GAPDH DNA ratios between TH>hUCP2 and control ies (Fig. 3A)(Neretti et al., 2009). We sought to further corroborate these ndingsby directly measuring mitochondrial volume density using transmis-sion electron microscopy (Medeiros, 2008). Mitochondrial quantica-tion of ultra-thin sections from indirect ight muscle bers revealed astatistically not signicant, 9% increase in mitochondrial volume frac-tion from indirect ight muscle bers of TH>hUCP2 ies as comparedto controls (Figs. 3BD). Taken together, these results suggest thatmolecular processes other than increased mitochondrial biogenesisare responsible for higher ATP content measured in TH>hUCP2 ies.

To assess mitochondrial function, we rst surveyed the expressionprole of representative subunits of each mitochondrial ETC complexand detected elevated expression levels of COI, a subunit for complexI and CG17856, a Drosophila homologue of ubiquinol-cytochrome-creductase, a key enzyme of complex III (Figs. 4AB), but not represen-tative subunits for complexes II, IV or V (Tiefenbock et al., 2010) (datanot shown). By performing quantitative real-time expression analy-sis, we found an average of 60% increase in COI expression levelsand 90% increase in CG17856 expression levels, respectively inTH>hUCP2 ies as compared to controls (Figs. 4AB). To correlate

140 R. Islam et al. / Neurobiology of Disease 46 (2012) 137146Fig. 2. An attenuated decline in age-dependent climbing ability and higher ATP levels mea-sured in TH>hUCP2 ies. (A and B) Attenuation in age-associated decline in climbingability in TH>hUCP2 ies. Longitudinal, negative geotaxis assays were performed onaged male (A) and female (B) ies and represented as averageSEM (N=12 trials/time point, **pb0.01, Student's t test). (C) An average of 44% higher steady state ATPlevels measured in 10-day-old TH>hUCP2 ies as compared to controls. Similar ATPlevels were detected between control and UAS-hucp2 ies (in the absence of the TH-Gal4 driver). Each value is represented as averageSEM (N=6, *pb0.05, Student's ttest). hUCP2: TH>hUCP2 ies; Control: TH>w1118 ies; UAS-hucp2: ies with the

UAS-hucp2 insertion only (in the absence of the TH-Gal4 driver).Fig. 3. No increased mitochondrial biogenesis detected in TH>hUCP2 ies. (A) No differ-ence in mitochondrial DNA/nuclear DNA ratio is detected between TH>hUCP2 iesand controls. Each bar represents averageSEM. N=6. (BD) Comparable mitochon-drial volume fractions are determined in the indirect ight muscle from control andTH>hUCP2 ies. Representative micrographs of ultra-thin sections of indirect ightmuscle from control (B) and TH>hUCP2 (C) ies, and quantication of mitochondrialvolume fractions (D) are shown. Mitochondrial volume fractions of each samplewere determined as described in Materials and Methods. Each value representsmeanS.E.M. (N=4 individual ies with eight elds from each y analyzed). Scale

bar=2 micrometer. Arrows indicate mitochondria.

in4067RNAb0.0an) mest)

141R. Islam et al. / Neurobiology of Disease 46 (2012) 137146transcript levels with the activity of those two complexes, we nextmeasured the NADH-dependent mitochondrial complex I activityand found an average of 61% increase in complex I activity in mito-chondria isolated from TH>hUCP2 ies as compared to controls(Fig. 4C). Interestingly, no signicant increase in complex III activityis measured in TH>hUCP2 as compared to control ies (Fig. 4D).Those results demonstrate that mitochondrial complex I activity isspecically and positively modulated in TH>hUCP2 ies.

Mammalian PGC-1 is a transcriptional co-activator regulating

Fig. 4. Transcript levels and activity of mitochondrial complexes I and III are modulatedTH>hUCP2 ies reveals an average of 60% increase in the expression level of COI (CG3pression level of CG17856, a subunit of mitochondrial complex III (B) as compared todetected in control ies was dened as 1.0. Each bar represents meanS.E.M. N=6, *presults using GAPDH or Tubulin as reference genes were obtained (data not shown). (Ccomplex I activity (C) whereas no signicant change is detected in complex III activity (Dtrol mitochondria. Each bar represents averageSEM. N=812, *pb0.05 (Student's t tmany metabolic programs including mitochondrial biogenesis andfunctions (Lustig et al., 2011). Recently, the Drosophila PGC-1 homo-logue, Spargel has been characterized to play an important role inmito-chondrial oxidative phosphorylation and insulin signaling but notmitochondrial biogenesis (Gershman et al., 2007; Tiefenbock et al.,2010). To investigate whether Spargel was involved in the increasedmitochondrial function measured in TH>hUCP2 ies, we assessed theexpression level of Spargel.Wedetected a signicant increase in Spargeltranscript levels in TH>hUCP2 ies where a 3.4 fold increase in Spargelexpression levels in heads (Fig. 5A) and a 2.3 fold increase in thoracesweremeasured as compared to control ies (Fig. 5B). Those results sug-gest that an overall up-regulation of Spargel present in TH>hUCP2 iesmay be responsible for increased mitochondrial function detected inthose ies. To further validate a role of Spargel in TH>hUCP2 ies, weexamined the expression pattern of a Spargel target gene, Tfam. UsingRNA isolated from larval fat bodies, Tfam was previously shown asone of the genes down-regulated in a Spargelmutant harboring a signif-icant reduction in Spargel transcripts (Tiefenbock et al., 2010). By per-forming quantitative expression analysis, we detected an average of63% increase in Tfam transcripts in TH>hUCP2 ies as compared to con-trols (Fig. 5C). Taken together, these results provide strong evidence in-dicating a functional involvement of Spargel and Tfam in modulatingmitochondrial activity in TH>hUCP2 ies.

Less ROS accumulation, heightened resistance to rotenone-inducedlethality and extended life span in TH>hUCP2 ies

Oxidative damage in the dopaminergic areas of the brain wasdetected in rats chronically exposed to rotenone (Betarbet et al.,2000; Sherer et al., 2003). To understand whether protection fromrotenone-induced neurotoxicity detected in TH>hUCP2 ies wasassociated with decreased oxidative damage in those ies, we mea-sured endogenous ROS levels in TH>hUCP2 and control ies. Asshown in Fig. 6A, a 21% lower endogenous ROS accumulation isdetected in TH>hUCP2 ies as compared to controls. These resultsare consistent with our ndings that TH>hUCP2 ies survive betterwhen exposed to rotenone (Figs. 6BC) or paraquat (data notshown), two agents known to generate ROS and oxidative damage

TH>hUCP2 ies. (A and B) Quantitative real-time PCR analysis of RNA isolated from), a subunit of mitochondrial complex I (A) and an average of 90% increase in the ex-isolated from control ies (Neretti et al., 2009). The expression level of target gene

5 (Student's t test). The housekeeping gene rp49 was used as a reference gene. Similard D) Mitochondrial complex activity assays reveal a 61% increase in NADH-dependenteasured in mitochondria isolated from TH>hUCP2 transgenic ies as compared to con-. hUCP2: TH>hUCP2 ies; Control: TH>w1118 ies.in vivo.As mentioned above, it is likely that systemic consequences in-

cluding increased mitochondrial function, elevated steady-state ener-gy levels, a minimal age-associated decline in motor activity and lessendogenous ROS accumulation measured in TH>hUCP2 ies are theresult of hucp2 expression in DA neurons and epidermis. To under-stand whether these positive physiological outcomes may be alsobenecial in the aging process leading to extended longevity, we per-formed survivorship studies of those ies. As shown in Figs. 7AB, amoderate but statistically signicant, 16% and 23% extension in medi-an life span in male and female TH>hUCP2 ies, respectively wasmeasured. Importantly, analysis of mortality rates reveals a slowerrate of aging of TH>hUCP2 ies as compared to genetically matchedcontrol ies (Figs. 7CD). A full statistical analysis of two independentlife span trials is shown in Supplemental Table 1.

Discussion

Successful recapitulation of key features of PD including motor de-ciency and DA neuron loss by exposing D. melanogaster to mitochon-drial toxins such as rotenone and paraquat makes it feasible toidentify cellular processes involved in PD associated pathogenesis inthis genetic model system (Chaudhuri et al., 2007; Coulom andBirman, 2004). As mitochondrial dysfunction has been recognized asan early step in DA neurodegeneration in both sporadic and heredi-tary forms of PD, defects in energy metabolism are likely to contributeto the pathogenesis of PD. Therefore, molecular approaches aimed atprotecting mitochondria from PD-associated toxins and improvingenergy metabolism should have a positive impact on halting the

142 R. Islam et al. / Neurobiology of Disease 46 (2012) 137146progression of PD. Our current study demonstrates a neuroprotectiverole of hucp2 expression in y DA neurons where rotenone-inducedDA neurodegeneration, head dopamine depletion and the subsequentmotor impairment are attenuated. We detect an up-regulation ofDrosophila PGC-1 homologue Spargel in TH>hUCP2 ies suggestinga role of this molecule in modulating mitochondrial function inthose ies. While a direct involvement of Spargel in neuroprotectionhas not been demonstrated, mammalian PGC-1 is emerging as acritical component in protecting against neurodegeneration. For ex-ample, PGC-1 knock-out mice, while metabolically compromisedas expected, display vacuolar degeneration of the striatum, an areatypically affected in Huntington's disease (Rona-Voros and Weydt,2010). Furthermore, PGC-1 knock-out mice are shown to be moresensitive to MPTP-induced DA neuron loss (St-Pierre et al., 2006). Inlight of our ndings, it is conceivable that activation of Spargel is in-volved in DA neuroprotection against rotenone by maintaining ener-gy homeostasis in those neurons. Supporting this notion is ourndings of an up-regulation of Tfam in TH>hUCP2 ies. Interestingly,while Tfam has been shown to be up-regulated by PGC-1 overex-pression and important for mitochondrial DNA maintenance andtranscription (Gleyzer et al., 2005), a recent study has demonstrateda protective role against MPP+ in Tfam overexpressing SH-SY5Y

Fig. 5. An up-regulation of Spargel and Tfam in TH>hUCP2 ies. Quantitative real-time PCRanalysis of RNA isolated from adult tissues reveals an average of 3.4 higher expressionlevel of Spargel in the heads (A) and an average of 2.3 higher expression level of Spargelin the thoraces (B) of TH>hUCP2 ies as compared to controls. (C) An increase of 63% inTfam transcript levels was detected in TH>hUCP2 ies as compared to controls. The ex-pression level of each target gene (Spargel or Tfam) detected in control ies was denedas 1.0. Each bar represents meanS.E.M. N=6, *pb0.05, **pb0.01 (Student's t test).The housekeeping gene rp49 was used as a reference gene. Similar results using GAPDHas the reference gene were obtained (data not shown). hUCP2: TH>hUCP2 ies; Control:TH>w1118 ies.neuroblastoma cells (Piao et al., 2011). Therefore, it is tempting topostulate that Tfam up-regulation is involved in the neuroprotectiveeffect in TH>hUCP2 ies. However, additional studies will be neces-sary to denitively establish a mechanistic relationship betweenhUCP2 activity and transcriptional activation of Spargel and Tfamleading to neuroprotection of DA neurons against mitochondrialtoxins.

Our transgenic model system allows us to investigate additionalphysiological effects associated with hucp2 expression under thecontrol of the TH promoter, active in DA neurons and epidermis. Wend that the systemic energy metabolism is better preserved inTH>hUCP2 ies in the presence of rotenone treatment. This apparentadaptation in energy production is physiologically signicant as it isalso associated with an overall better climbing ability measured inTH>hUCP2 ies under normal growing conditions. As steady stateATP levels measured in those ies were largely from mitochondriarich, thoracic muscle tissue, DA and epidermal expression of hucp2cannot be entirely responsible for changes in ATP levels. Therefore,we propose that a cell-non-autonomous mechanism may be involvedin modulating ATP levels in TH>hUCP2 ies. As UCP2 is known to un-couple substrate oxidation and ATP phosphorylation, our results

Fig. 6. Decreased endogenous ROS accumulation and increased resistance to rotenoneexposure in TH>hUCP2 ies. (A) A 21% decrease in ROS accumulation is measured inTH>hUCP2 ies as compared to controls. Each bar represents meanS.E.M. N=7,**pb0.01 (Student's t test). (B and C) Rotenone induced lethality in control ies is atten-uated in TH>hUCP2 ies. Ten-day-old ies were treated with 500 M rotenone and sur-vival assays were conducted. Consistent results are measured in both male (B) andfemale (C) ies. Each value represents meanS.E.M. (N=912 vials with 20 ies/vial).All differences measured between control and hUCP2 ies are statistically signicant(**pb0.01, Student's t test). hUCP2: TH>hUCP2 ies; Control: TH>w1118 ies.

A) adianle T(C)TH>aysnic(C an; Co

143R. Islam et al. / Neurobiology of Disease 46 (2012) 137146Fig. 7. An extended life span in TH>hUCP2 ies. (A and B) Survivorship curves for male (37 days for control ies and 43 days for TH>hUCP2 ies, respectively. (B) Female merank analysis shows a statistically signicant increase of 16% in median life span in mato controls (see Supplemental Table 1). (C and D) Age-specic mortality analysis of malerate is plotted. hUCP2: TH>hUCP2 ies; Control: TH>w1118 ies.An extended life span incontrol ies are shown. (A) Male median life spans are 37 days for control ies and 43 dand 43 days for TH>hUCP2 ies, respectively. Log rank analysis shows a statistically sigincrease in female TH>hUCP2 ies as compared to controls (see Supplemental Table 1).ies is shown. Natural log (Ln) of the mortality rate is plotted. hUCP2: TH>hUCP2 iessuggest an intriguing possibility that hUCP2 activity in a subset ofneurons and epidermis may release a stress signal in the form of ametabolite or cytokine. This stress signal is then perceived in a cell-non-autonomous manner that in turn triggers transcriptional activa-tion of Spargel leading to systemic enhancement in mitochondrialfunction and energy homeostasis. A recent study in C. elegans hasshown that broad physiological processes such as aging can bemodulated in a cell-non-autonomous fashion by manipulating mito-chondrial functions (Durieux et al., 2011). In this study, it is shownthat perturbation of a mitochondrial ETC component, cytochrome coxidase-1 subunit Vb/COX4 (cco-1) in the nervous system is per-ceived and responded to by an unfolded protein process (UPR) in-volved in mitochondrial stress responses in the intestine (Durieuxet al., 2011). It is postulated by the authors that a mitokine maybe responsible for transmitting the signal leading to cell-non-autonomous longevity effects although neither the nature of this pro-posed mitokine nor its specic mode of action is yet known (Durieuxet al., 2011). Our ndings present a novel aspect of a similar scenariowhere modulation of hUCP2 activity under the control of the TH pro-moter leads to long-range physiological adaptations in energy metab-olism. Additional studies are needed to further test this hypothesis.

Interestingly, an elevated ATP content measured in TH>hUCP2ies is not associated with increased mitochondrial biogenesis asevidenced by the lack of signicant changes in mitochondrial DNAcontent and mitochondrial volume fraction. In contrast, we detecttranscriptional activation of subunits of mitochondrial complexes Iand III with only increased mitochondrial complex I activity. Accom-panying those results is an up-regulation of Spargel. While its mam-malian counterpart PGC-1 is a major regulator for mitochondrialbiogenesis and respiration in brown adipose tissue (Puigserver etal., 1998), muscle (Lin et al., 2002) and liver (Herzig et al., 2001),Spargel has been recently shown to be essential in mitochondrialfunction but not mass (Tiefenbock et al., 2010). As reported bynd female (B) TH>hUCP2 and control ies are shown. (A) Male median life spans arelife spans are 35 days for controls and 43 days for TH>hUCP2 ies, respectively. LogH>hUCP2 ies and an average of 23% increase in female TH>hUCP2 ies as comparedand female (D) control and TH>hUCP2 ies is shown. Natural log (Ln) of the mortalityhUCP2 ies. (A and B) Survivorship curves for male (A) and female (B) TH>hUCP2 andfor TH>hUCP2 ies, respectively. (B) Female median life spans are 35 days for controlsant increase of 16% in median life span in male TH>hUCP2 ies and an average of 23%d D) Age-specic mortality analysis of male (C) and female (D) control and TH>hUCP2ntrol: TH>w1118 ies.Tiefenbock et al., despite a broad down-regulation of multiple genesencoding mitochondrial ETC complexes detected in the larval fatbody of a Spargel mutant, only complex II activity is affected(Tiefenbock et al., 2010). Our results demonstrating an increasedcomplex I activity in TH>hUCP2 adult ies are consistent with the no-tion that Spargel coordinates mitochondrial function whereas theexact mechanism remains to be determined. In cultured cells, milduncoupling via chemical uncouplers was previously shown to inducePGC-1 and PGC-1 expression and mitochondrial biogenesis tomaintain ATP levels and cell survival (Rohas et al., 2007). Our results,on the other hand, provide novel evidence that Spargel may be a cru-cial molecular switch in regulating global energy metabolism by sens-ing energy cues as the consequence of hUCP2 action in DA neuronsand epidermis. A role of UCP2 in energy adaptation has been shownin neurons (Diano et al., 2003) and glia (Besson et al., 2010). In atransgenic model harboring ubiquitous hucp2 expression, increasedneuronal survival following induction of motor seizures is associatedwith increased ATP levels (Diano et al., 2003). More recently, hucp2expression in Drosophila glia is shown to rescue the mutant Huntingtinprotein (mHtt) induced motor impairment and lethality suggestingan energy-dependent function in glia (Besson et al., 2010). Our currentreport provides rst evidence that expression of hucp2 under the con-trol of the TH promoter leads to physiological adaptations in energymetabolism.

We show that TH>hUCP2 ies accumulate less endogenous ROS andare more resistant to environmental oxidants than controls. A role ofSpargel in ROS metabolism has not been examined (Tiefenbock et al.,2010). On the other hand, PGC-1 and PGC-1 have been demonstrat-ed as potent ROS suppressors (St-Pierre et al., 2006). In cultured striatalcells, PGC-1 is shown to protect cells from oxidative stressors by in-ducing the expression of several ROS-detoxifying enzymes (St-Pierreet al., 2006). Furthermore, PGC-1 has recently been shown to be in-volved in endothelial migration through regulation of H2O2 levels thus

144 R. Islam et al. / Neurobiology of Disease 46 (2012) 137146rmly placing this transcriptional co-activator as a ROSmodulator func-tional in several biological systems (Borniquel et al., 2010). Based on ourresults, we postulate that while increasedmitochondrial activity is pre-sent in TH>hUCP2 ies and could lead to higher ROS production, activa-tion of Spargel may act to suppress endogenous ROS accumulation.

Taken together, we have expressed hUCP2 in y DA neurons andepidermis. Our study reveals a neuroprotective effect of hUCP2 in aDrosophila sporadic PD model and broad, favorable physiological out-comes including enhanced energy metabolism, a decrease in age-associated decline in locomotor activity, decreased endogenous ROSlevels and extended longevity. These ndings have led us to proposethat those physiological outcomes measured in TH>hUCP2 ies arethe result of both cell-autonomous and cell-non-autonomous re-sponses involving transcriptional activation of Spargel.

Materials and Methods

Generation and maintenance of y lines

The TH-Gal4 driver line was obtained from the Bloomington StockCenter (stock# 8848) (Friggi-Grelin et al., 2003). The homozygousUAS-hucp2 y line was previously shown to produce a functionalhUCP2 protein when targeted to the adult nervous system (Fridell etal., 2005). The UAS-hucp2 stock was backcrossed into the w1118 geneticbackground for 10 generations as described previously (Fridell et al.,2005). All y stocks were maintained in a humidied, temperature-controlled incubator with 12 h on/off light cycle at 25 C on standardcorn meal/yeast/sucrose/agar diet.

Rotenone and paraquat treatment of adult ies

Ten-day-old TH>hUCP2 and TH>w1118 ies were starved on 1%agar containing vials for 6 h before subjected to rotenone or paraquattreatment. For rotenone treatment, ies were transferred to vials con-taining 1% agar mixed with 5% sucrose and 500 M rotenone (Coulomand Birman, 2004). For paraquat treatment, ies were transferred tovials containing lters soaked with 10 mM paraquat in 5% sucrose(Fridell et al., 2005).

Head dopamine measurements by HPLC

Ten-day-old TH>hUCP2 and TH>w1118 ies were treated with500 M rotenone for four days. Fly heads were collected on dry iceand immersed in 60 l of chilled 0.1 M perchloric acid. Three heads/sample were sonicated briey and spun at 14,000 rpm at 4 C for20 min. The supernatant was injected to a HPLC system for dopamineanalysis. Briey, 15 l supernatant was isocratically eluted through an804.6 mm C18 column (ESA Inc., Chelmsford, MA) with a mobilephase containing 100 mM LiH2PO4, 1.5 mM 1-octanesulfonic acidand 7% (v/v) methanol and detected by a dual-channel CoulochemII electrochemical detector (ESA Inc., Chelmsford, MA) set as Channel1 at 50 mV and Channel 2 at 340 mV. Running time was 15 min anddopamine was eluted around 5 min. Synthetic dopamine (Sigma-Aldrich) was used as the external standard for calibration and calcula-tion. Dopamine levels are expressed by picograms per y head.

Negative geotaxis assays

Ten-day old ies were subjected to negative geotaxis assays es-sentially as previously described (Coulom and Birman, 2004; Feanyand Bender, 2000). Briey, twenty male or female TH>hUCP2 andcontrol ies were separated and transferred to an empty vial tapedto another empty vial end to end. Flies were gently tapped to the bot-tom of the vials and allowed to climb up within 1 min. At the end ofthe 1-minute interval, ies that reached the top vial (after climbing

3.7 in. in height) were counted and percentages of climbing activitywere determined as percentages of ies that reached the top vial.Three replicate samples per each genotype were included in each ex-periment with three trials of each sample performed. At least two in-dependent experiments were performed. Age-dependent negativegeotaxis was performed with twenty male or female TH>hUCP2and control ies at age 10, 15, 20, 25, 30, 35 and 40 days. To deter-mine the climbing ability of ies treated with mitochondrial toxins,similar negative geotaxis assays were performed as described above.Ten-day old ies were separated by sex and the baseline climbing ac-tivity (day 0) was recorded prior to transferring ies to 10 mM para-quat or 500 M rotenone containing agar/sucrose vials as describedabove. Negative geotaxis assays were then performed everydaythereafter for seveneight days. Three independent experimentswere performed for all negative geotaxis assays.

Immunouorescent staining of whole-mount adult brains

Ten-day-old TH>hUCP2 and TH>w1118 ies were treated with500 M rotenone for three days. Untreated TH>GFP ies were used asreference for visualizing DA neuron clusters (data not shown). Whole-mount immunouorescent staining of adult brains was performed es-sentially as described (Wang et al., 2006). Briey, following xation in4% paraformaldehyde, adult brains were dissected and permeabilizedwith 1% Triton/PBS, blocked with 5% normal goat serum and immuno-probedwith an anti-TH antibody at 1:1000 (ImmunoStar Inc.) followedby immunostaining with a secondary antibody conjugated with TRITCat 1:1000 (Jackson ImmunoResearch). The immunostained brainswere mounted on cover slips for confocal microscopy. Z-series imagesat 1-m intervals were captured with 40 objectives and TH+ DA neu-rons were quantied with a blind method as described previously(Fridell et al., 2009). Essentially, each experiment involved two peoplewhere one person performed the staining and the other read the slides,which have been numerically blinded by the rst person. The numberof DA neurons in each major cluster was averaged from at least vebrains from each genotype per experiment. Three independent experi-ments were performed.

Quantitative real-time and semi-quantitative RT-PCR expression analysis

Total RNAwas isolated from heads or thoraces of 10-day-old femalesraised on standard diet using the TRIzolmethod (Invitrogen), and subse-quent cDNA and real-time RT-PCR experiments were performed as de-scribed previously (Fridell et al., 2009). Semi-quantitative PCR analysiswas performed as described previously (Fridell et al., 2005). Three inde-pendent RNApreparationswith triplicates in each experimentwere usedto derive the mean ratios of target gene expression against referencegenes rp49, GAPDH or tubulin (Fridell et al., 2009). The primer sequencefor detecting the expression of CG9809/Spargel, COI (CG34067)CG17856, rp49, GAPDH and tubulin was described previously (Nerettiet al., 2009). The following primers were used to detect target genesTfam (CG4217), Scs-fp (CG17246, complex II subunit), RFeSp (CG7361,complex III subunit), CoVa (CG14724, complex IV subunit) and Blw(CG3612, complex V subunit): Tfam-F, 5-CACCTCGACGGTGGTAATCT-3; Tfam-R, 5-AAGACCCTGGAGGAGCAGTT-3; ScS-Fp-F, 5-CAGCG-CAGTTACCACATCACA-3, ScS-Fp-R, 5-TTCGATATCTTGTCCGGATTCG-3;RFeSp-F, 5-CGGAGGGCAAGTCGGTTAC-3, RFeSp-R, 5-TGCGGTGGCG-GATGA-3; CoVa-F, 5-CGCGTCAACGACATTGCA-3, CoVa-R, 5-TTGG-TCGCCGCACTTGT-3; Blw-F, 5-TCCAGGCCGATGAGATGGT-3, Blw-R,5-TGTCGGGCTCCAAGTTAAGG-3 (Tiefenbock et al., 2010).

ATP measurements

Total steady-state ATP levels from heads and thoraces of femaleies were measured using the ATP Determination Kit (A22066, Invi-trogen) as previously described (Park et al., 2006). The abdomen of

each y was removed to eliminate potential ATP contribution from

145R. Islam et al. / Neurobiology of Disease 46 (2012) 137146eggs. Total ATP levels were determined using a Luminometer(FLx800, BioTek) according to the manufacturer's instructions andnormalized to total protein in each sample (Park et al., 2006).

Transmission electron microscopy and determination of mitochondrialvolume fraction

Each y was immobilized by a brief exposure to low temperature(0 C) during which head, wings, legs and abdomen were removedand discarded. A 0.1 mm minutien pin was inserted just under theventral cuticle of the remaining thorax and a slit was cut throughthe adjacent cuticle to improve penetration of solutions. Embedmentof thoraces included the following steps: aldehyde xation (2% glu-taraldehyde, 1% paraformaldehyde, 0.1 M HEPES, 0.120 M NaCl,0.0015 M MgCl2, pH 7.3) for 16 h at 4 C; buffer wash (0.1 M HEPES,0.120 M NaCl, 0.0015 M MgCl2, pH 7.3) three times for 1 h each at25 C; osmication (2% OsO4 in 0.1 M HEPES, 0.120 M NaCl, 0.0015 MMgCl2), 1.5 h at 25 C; water wash three times for 40 min each at25; 50% ethanol for 45 min at 25; 70% ethanol, overnight at 4 C,95% ethanol for 30 min at 25 C, 100% ethanol twice for 30 min at25 C, propylene oxide (PO) 20 min at 25 C, 1:1 PO: medium hard-ness epoxy resin for 2.5 h at 25 C; 1:3 PO: resin for 16 h at 25 C;100% resin for 9.5 h (with one change); embedment in at moldsand polymerization at 60 C for 48 h; All steps at 25 C were carriedout on a rolling mixer (Glauert, 1991). Blocks were positioned andto allow sagittal sectioning of the entire thorax. The block was thenmounted in an ultramicrotome and cut until the six dorsal median in-direct ight muscles could be seen in methylene blue stained 1 msections. Finally, ultrathin (~100 nm) sections were cut and stainedwith 2% uranyl acetate and Sato's lead citrate. Ultrathin sectionswere examined in an FEI Tecnai 12 Biotwin transmission electron mi-croscope equipped with an AMT XR40 4 Megapixel CCD camera. Foreach of eight thoraces (four TH>hUCP2 and four control) eight im-ages were recorded of 16.416.4 m elds randomly distributedalong the length of the second or third dorsal median indirect ightmuscle. A three micrometer grid was superimposed on each image,and the number of intersections falling within mitochondria in eachimage was determined. The mitochondrial volume fraction was de-termined as the ratio of mitochondrial intersections to total intersec-tions (Medeiros, 2008). A two-sample t-test was applied to test forstatistical signicance.

Determination of mitochondrial DNA content

Mitochondrial DNA content was determined by the ratio of thegene for cytochrome oxidase subunit I (COI) to a nuclear geneGAPDH as previously described (Neretti et al., 2009). Total DNA wasisolated from 10-day-old ies in the presence of proteinase K (Saikiet al., 1988). Real-time quantitative PCR experiments were then per-formed using a SYBR Green Master Mix (Biorad), 7500 Fast Real-TimePCR System (Biorad), and gene-specic primers for COI and GAPDH asdescribed previously (Neretti et al., 2009). The two-step PCR cyclecomprised of denaturation at 95 C (15 s) and annealing and exten-sion at 60 C (1 min) was carried out for 30 cycles. Ten biologicalsamples were collected with triplicates performed for each sample.

Mitochondrial complex activity assays and hydrogen peroxidemeasurements

Mitochondrial respiratory complex I and III activities inTH>hUCP2 and control ies were determined essentially as previous-ly described (Neretti et al., 2009). Briey, mitochondria were isolatedfrom heads and thoraces of 10-day-old ies as previously described(Fridell et al., 2005). Isolated mitochondria were then sonicated andsubjected to activity assays as previously described (Neretti et al.,

2009).Hydrogen peroxide accumulation in isolated mitochondria wasmeasured under normal respiration conditions using the AmplexRed kit (Molecular Probes) and determined using a spectrophotome-ter at absorption maximum of 563 nm as previously described(Fridell et al., 2005).

Life span and stress resistance studies

To perform life span studies, homozygous virgins bearing the UAS-hucp2 transgene and control w1118 female virgins were crossed to TH-Gal4 drivermales to obtain TH>hUCP2 and TH>w1118 ies. TH>hUCP2and TH>w1118 ies were maintained on standard corn meal/yeast/sucrose/agar diet and passed to fresh vials every other day and num-ber of dead ies recorded (Fridell et al., 2005). Two independenttrials were performed. Numbers of ies included in each trial arelisted in Supplemental Table 1.

Both rotenone and paraquat resistance assays were conducted asdescribed previously (Fridell et al., 2005). Briey, 10-day-oldTH>hUCP2 and TH>w1118 ies raised on standard diet were placed invials containing 10 mM paraquat or 500 M rotenone dissolved in 5%sucrose/1% agar and the number of dead ies counted every day.Three independent experiments were performed. Each experimentused four to ve vials with 20 males or 20 females in each vial.

Statistical analysis

Statistical analysis for independent life span trials was performedusing log-rank test (GraphPad). Age-specic mortality is calculatedas Ln(px) where px is the probability of surviving to age x. Resultsfor all other assays were analyzed using Student's t test.

Supplementary materials related to this article can be foundonline at doi:10.1016/j.nbd.2011.12.055.

Funding

This work was supported by grants from the NIA to Y-W.C.F(AG21068, AG31086).

Acknowledgments

The authors wish to thank Drs. Ping Zhang and Daewoo Lee forcritical review of the manuscript, Ritvik Dubey for assistance in lifespan data analysis, Joseph Cichocki for assistance in initial negativegeotaxis assays and members of the Fridell lab for helpful discussion.We also thank Stephen Daniels of the Electron Microscopy Laboratoryfor his help and advice in developing the EM protocols.

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A neuroprotective role of the human uncoupling protein 2 (hUCP2) in a Drosophila Parkinson's Disease modelIntroductionResultshucp2 expression in DA neurons protects flies from rotenone-induced DA neuron death, head dopamine depletion, locomotor imp...TH&gt/;hUCP2 flies are more resistant to age-associated decline in climbing activity and appear to maintain enhanced energy...Increased mitochondrial function in THhUCP2 fliesLess ROS accumulation, heightened resistance to rotenone-induced lethality and extended life span in THhUCP2 flies

DiscussionMaterials and MethodsGeneration and maintenance of fly linesRotenone and paraquat treatment of adult fliesHead dopamine measurements by HPLCNegative geotaxis assaysImmunofluorescent staining of whole-mount adult brainsQuantitative real-time and semi-quantitative RT-PCR expression analysisATP measurementsTransmission electron microscopy and determination of mitochondrial volume fractionDetermination of mitochondrial DNA contentMitochondrial complex activity assays and hydrogen peroxide measurementsLife span and stress resistance studiesStatistical analysis

FundingAcknowledgmentsReferences