UCP4A protects against mitochondrial ... - The FASEB Journal

The FASEB Journal article fj.14-255802. Published online August 21, 2014.

The FASEB Journal • Research Communication

UCP4A protects against mitochondrial dysfunction and degeneration in pink1/parkin models of Parkinson’s disease Kai Wu,*,1 Jia Liu,*,†,1 Na Zhuang,*,‡,1 and Tao Wang*,2 *National Institute of Biological Sciences, Beijing, China; †College of Life Sciences, Beijing Normal University, Beijing, China; and ‡School of Life Sciences, Tsinghua University, Beijing, China Genetic mutations in parkin or pink1 are the most common causes of familial Parkinson’s disease. PINK1 and Parkin are components of a mitochondrial quality control pathway that degrades dysfunctional mitochondria via autophagy. Using a candidate gene approach, we discovered that overexpression of uncoupling protein 4A (ucp4A) suppresses a range of pink1 mutant phenotypes, including male sterility, locomotor defects, and muscle degeneration that result from abnormal mitochondrial morphology and function. Furthermore, UCP4A overexpression in pink1 mutants rescued mitochondria-specific phenotypes associated with mitochondrial membrane potential, production of reactive oxygen species, resistance to oxidative stress, efficiency of the electron transport chain, and mitochondrial morphology. Consistent with its role in protecting mitochondria, UCP4A rescued mitochondrial phenotypes of parkin mutant flies, as well. Finally, the genetic deletion of ucp4A resulted in increased sensitivity to oxidative stress, a phenotype that was enhanced by the loss of PINK1. Taken together, these results indicate that UCP4A prevents mitochondrial dysfunction and that modulation of UCP activity protects cells in a situation relevant for human Parkinson’s disease.—Wu, K., Liu, J., Zhuang, N., Wang, T. UCP4A protects against mitochondrial dysfunction and degeneration in pink1/parkin models of Parkinson’s disease. FASEB J. 28, 000 – 000 (2014). www.fasebj.org

ABSTRACT

Key Words: ROS 䡠 ETC 䡠 Drosophila Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic (DA) neurons (1–3). Although most cases are sporadic, inheritable genetic Abbreviations: BSA, bovine serum albumin; DA, dopaminergic; DCFH-DA, 2=,7=-dichlorofluorescein diacetate; DHE, dihydroethidium; DL, dorsolateral; ETC, electron transport chain; HTRA2, HtrA serine peptidase 2; LRRK2, leucine-rich repeat kinase 2; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PINK1, PTEN-induced putative kinase 1; ROS, reactive oxygen species; TEM, transmission electron microscopy; TH, tyrosine hydroxylase; UCP, uncoupling protein; UCP4A, uncoupling protein 4A; WT, wild type 0892-6638/14/0028-0001 © FASEB

defects associated with familial cases have provided unique insights into the pathogenesis of PD. Genes linked to heritable forms of PD suggest that its causes include mitochondrial dysfunction and aberrant protein degradation. However, the precise mechanism of PD’s pathogenesis remains unclear, and current treatments have failed to slow the disease’s progression. Mitochondria are the powerhouses of the cell (4), and mitochondrial dysfunction, which can be caused by genetic or environmental factors, depletes cellular energy, disrupts cellular homeostasis, and can lead to cell death. Several lines of evidence suggest that mitochondrial dysfunction plays a role in PD’s pathogenesis (5). Patients with PD have mitochondrial abnormalities, including defects in oxidative phosphorylation, defects in mitochondrial DNA, accumulation of reactive oxygen species (ROS), and dissipation of mitochondrial membrane potential (6). In addition, MPTP and rotenone [inhibitors of complex I in the mitochondrial electron transport chain (ETC)] are associated with sporadic PD and cause clinical features that resemble PD in animal models (7, 8). More conclusively, disruption of the mitochondrial quality-control genes parkin, which encodes an E3 ubiquitin ligase, and PTENinduced putative kinase 1 (pink1), which encodes a mitochondrially targeted protein kinase, results in autosomal recessive PD (9 –11). PINK1 and Parkin participate in the selective autophagy of damaged mitochondria (12–17). Losses in mitochondrial membrane potential, which characterize dysfunctional mitochondria, stabilize PINK1 on the mitochondrial outer membrane. PINK1 then recruits and activates Parkin, thereby triggering mitochondrial engulfment and degradation through autophagy (18 –23). Uncoupling proteins (UCPs) are mitochondrial anion carriers that localize to the mitochondrial inner 1

These authors contributed equally to this work. Correspondence: National Institute of Biological Sciences, 7 Park Road, Zhongguancun Life Science Park, Changping District, Beijing 102206, China. E-mail: wangtao@ nibs.ac.cn doi: 10.1096/fj.14-255802 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2

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membrane and can disrupt the ETC-generated proton gradient (24). Besides modulating thermogenesis, UCP-mediated decreases in the mitochondrial membrane potential can lower ROS production. Mammalian UCP2 exhibits neuroprotective activity in response to a variety of insults, such as epilepsy and stroke (25, 26). Furthermore, UCP2 overexpression prevents MPTP-induced damage to DA neurons, and loss of ucp2 increases oxidative damage in DA neurons (27–29). Mitochondrial uncoupling has been observed in models of PD that involve mutations in leucine-rich repeat kinase 2 (LRRK2) and HtrA serine peptidase 2 (HTRA2) (30, 31). In addition, mitochondrial uncoupling makes select populations of neurons more vulnerable to degeneration in PD (30 –32). We therefore asked whether Drosophila UCPs could modify pink1 or parkin mutations. Overexpression of UCP4A in pink1 or parkin mutants suppressed mitochondria degeneration, and flies lacking UCP4A were more vulnerable than the controls to oxidative stress.

MATERIALS AND METHODS

solution (ATP Assay Kit; Beyotime, Beijing, China), and luminescence was measured with a luminometer (EnSpire Multimode Plate Reader; PerkinElmer, Waltham, MA, USA). To measure mitochondrial ATP synthesis, pyruvate/malate was added to isolated fly mitochondria, and newly generated ATP was measured in a time series. Assay for oxidative stress resistance Flies that were 3 d old were raised on normal food for 1 d before they were transferred to food containing 20 mM paraquat or 5 mM rotenone. The flies were transferred to new food every 2 d. Fly survival was monitored over the next 220 h. At least 100 flies per genotype were analyzed. Isolation of mitochondria The isolation of fly mitochondria was performed as described previously (34). Briefly, thoraxes from flies that were 3–5 d old were gently homogenized in 1 ml of chilled mitochondrial isolation buffer (250 mM sucrose; 10 mM Tris, pH 7.4; and 0.15 mM MgCl2). The homogenate was centrifuged at 1000 g for 5 min at 4°C to remove debris, and then the supernatant was centrifuged again at 13,000 g for 10 min to pellet the mitochondria. Mitochondrial concentrations were determined by measuring total protein with the Bradford method.

Drosophila stocks The pink1B9 and park1 stocks are both genetic nulls (12, 14). Male pink1B9 and park1 flies were used for all experiments. Transgenic UAS-ucp4A, UAS-ucp4B, and UAS-ucp4C strains were generated by P-element-mediated transformation. The UAS-bmcp stock was a gift from Dr. Stephen Helfand (Brown University, Providence, RI, USA). The w1118, Act-Gal4, DaGal4, ucp4AG1388, and Tub-Gal4 flies were obtained from the Bloomington Stock Center (Indiana University, Bloomington, IN, USA). All flies were maintained at 25°C. Generation of transgenic flies and ucp4A deletion The EST clones RH64870, IP15246, and AT16588 were used to generate UAS-ucp4A, UAS-ucp4B, and UAS-ucp4C constructs, respectively. The constructs were individually injected into w1118 embryos, and transformants were identified on the basis of eye color. Imprecise excision of the P-element insertion line ucp4AG1388 (by introducing ⌬2–3 transposase) was used to generate ucp4A-deletion mutants. The ucp4A deletions were screened with PCR and confirmed with DNA sequencing. Climbing assays Climbing assays were performed as described, with modifications (33). Groups of five 3-d-old flies were transferred into climbing-ability test vials and incubated for 1 h at room temperature, to allow for environmental adaptation. The flies were then tapped to the bottom of the vial, and the time that it took for a median number of flies to climb 10 cm was recorded. For each genotype, ⱖ100 flies were tested, and the average climbing time (mean⫾sd) was calculated.

Measurement of ROS production The fluorescent probe dihydroethidium (DHE; Sigma-Aldrich, St. Louis, MO, USA) was used to measure superoxide production, and the cell-permeable probe 2=,7=-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) was used to measure hydrogen peroxide production. Briefly, muscles were dissected from flies and incubated in phosphate-buffered saline (PBS) with 20 ␮M DHE for 1 h or with 10 ␮M DCFH-DA for 20 min at room temperature in the dark. The tissues were then washed with PBS, and images were captured immediately with a confocal microscope (A1-R; Nikon, Tokyo, Japan). Phase-contrast microscopy and transmission electron microscopy (TEM) For light microscopic analysis of male germ lines, testes were dissected in PBS from 3-d-old male flies (to isolate spermatids during the onion stage) and imaged with a microscope (Axiovert 200; Zeiss, Göttingen, Germany) equipped with a ⫻40 phase-contrast objective. For TEM, testes or thoraxes were dissected from 3-d-old males and fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in PBS overnight at 4°C. After they were rinsed in PBS, the samples were postfixed in 1% OsO4 for 1 h in the dark at 4°C. The tissues were then dehydrated in an ethanol series and embedded in Epon reagent (Ted Pella, Inc., Redding, CA, USA). Tissue sections were cut (50 – 80 nm), stained with uranyl acetate and lead citrate, and examined by TEM (JEM-1400; JEOL, Tokyo, Japan). Images were acquired with a Gatan camera (model 832; Gatan, Pleasanton, CA, USA). TUNEL assay and immunostaining

ATP synthesis measurement To measure total ATP, 10 thoraxes from flies that were 3–5 d old were dissected and homogenized in extraction buffer. The homogenized supernatant was mixed with a luminescent 2

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The Click-iT TUNEL imaging assay kit (Invitrogen, Carlsbad, CA, USA) was used to detect apoptotic cells. Briefly, thoraxes were dissected from 3-d-old flies, fixed in 4% formaldehyde in PBS for 3 h at 4°C, permeabilized with 0.25% Triton X-100 in

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PBS for 20 min, washed with deionized water, incubated in TdT reaction cocktail for 1 h at 37°C, washed twice with 3% bovine serum albumin (BSA) in PBS for 2 min, and incubated with the Click-iT reaction cocktail for 30 min at room temperature in the dark. For the immunostaining assay, adult brains were dissected and stained with rabbit anti-TH antibody (1:200; Millipore, Billerica, MA, USA). Microscopic analysis was performed with an Eclipse Ni microscope (Nikon). Measuring oxygen consumption The rate of oxygen consumption was measured polarographically with a Clark-type oxygen electrode (Oroboros Instruments, Innsbruck, Austria) at 25°C, as described previously (35). Briefly, mitochondria were isolated from 3-d-old flies, and then mixed with 1 ml of respiratory buffer (250 mM sucrose, 15 mM K2HPO4, 15 mM HEPES, 2 mM EGTA, and 0.4% fatty-acid–free BSA, pH 7.2). The rate of oxygen consumption was measured in the presence of 0.25 mM ADP, 0.5 M pyruvate, and 0.05 mM malate. Total protein levels were determined by the Bradford method. Measurement of mitochondrial membrane potential Mitochondrial membrane potential was measured with the JC-10 reagent (Enzo Life Sciences, Farmingdale, NY, USA). Briefly, indirect flight muscles were dissected in PBS from 3-d-old flies and then stained with 100 nM JC-10 for 15–30 min. Confocal images were obtained with a Nikon A1 confocal microscope by monitoring fluorescence intensities at excitation/emission wavelengths of 490/525 nm and 540/590 nm. The ratio of fluorescence emissions at 525 and 590 was used for data analysis.

RESULTS UCP4A rescues pink1 mutant phenotypes The Drosophila genome contains 4 genes that encode UCPs: UCP4A, UCP4B, UCP4C, and brain mitochondrial carrier protein (BMCP; also known as dmUCP5) (36, 37). To test whether mild mitochondrial uncoupling ameliorates phenotypes caused by loss of PINK1, we overexpressed fly UCPs on the pink1B9 mutant background. Phenotypes associated with pink1 mutant animals include a crushed thorax, defective locomotor activity, and male sterility. Both crushed thorax and defective locomotor activity are the results of degeneration of indirect flight muscles, whereas male sterility is caused by mitochondrial and individualization defects in spermatids (13, 14). The crushed-thorax phenotype associated with the pink1 mutation was dramatically rescued by the expression of UCP4A (pink1B9; Tub⬎ucp4A: pink1B9; Tub-Gal4/UAS-ucp4A), whereas the effects of the other UCPs were not significant (Fig. 1A). Similarly, pink1-associated locomotion defects were suppressed by UCP4A, but not by the other UCPs, as UCP4A expression significantly restored the climbing abilities of pink1 mutant animals (Fig. 1B). Furthermore, TUNNEL staining of thoraxes showed that UCP4A expression reduced cell death within the indirect flight muscle of pink1 mutant flies (Fig. 1C). UCP4A PROTECTS AGAINST PARKINSON=S DISEASE

Finally, we used TEM to determine whether UCP4A affects mitochondrial morphology within pink1 mutant muscle fibers. In wild-type (WT) adults, muscle fibers and mitochondria were organized into parallel stripes, and the mitochondria had densely packed cristae (Fig. 1D). In contrast, muscle fibers from young pink1 mutants were disorganized and fragmented, and mitochondria had very few cristae (Fig. 1D). These muscle fiber and mitochondrial morphology phenotypes were nearly fully rescued by UCP4A overexpression (Fig. 1D). The pink1-null mutant males are sterile because of mitochondrial defects during spermatogenesis. We therefore asked whether UCPs could rescue this phenotype. Overexpression of UCP4A in pink1 mutants significantly suppressed male sterility, although expression of UCP4B or UCP4C also resulted in a few fertile males. During spermatogenesis, mitochondria within early spermatids aggregate and fuse, thereby forming a giant mitochondrion called the nebenkern (38). Phasecontrast microscopy of WT onion-stage spermatids revealed 2 adjacent spherical structures: the nucleus and the nebenkern (Fig. 1E). In pink1 mutant testis, spermatids were disorganized, and the nebenkerns were distorted and vacuolated, whereas the expression of UCP4A fully restored the morphology of pink1 mutant spermatids (Fig. 1E). Later in the spermatogenic process, after elongation and individualization, each spermatid consists of a central axoneme and a mitochondrial derivative (Fig. 1E). Within pink1 mutant cysts, spermatids exhibited disorganization, defects in mitochondrial morphology, and mitochondrial loss. UCP4A expression restored spermatid organization and mitochondrial morphology at this stage, as well (Fig. 1E). DA neurodegeneration is one of the major characteristics of PD, and the pink1 mutant flies exhibit extensive DA neuron degeneration (14). Using anti-tyrosine hydroxylase (TH) staining, a small but significant loss of DA neurons was detected in the pink1 mutant brain at 22 d of age, especially in the dorsolateral (DL1) cluster (Supplemental Fig. S1). This reduction in DA neurons in the pink1 mutant animals was suppressed by expression of UCP4A as well (Supplemental Fig. S1). UCP4A protects mitochondria function in pink1 mutant animals In multiple systems, the loss of PINK1 impairs mitochondrial respiration and ATP synthesis (34). We reasoned that UCP4A would rescue the pink1 mutant phenotype by improving mitochondrial function through mild uncoupling. First, we measured steadystate ATP levels to determine whether the expression of UCP4A increases energy production of pink1 mutant cells. ATP depletion was less pronounced in the pink1; Tub⬎ucp4A flies than in the pink1 mutant animals (Fig. 2A). To test whether this increase in steady-state ATP results from improved mitochondrial ATP synthesis, we measured respiration and ATP synthesis in mitochondria isolated from pink1 and pink1;Tub⬎ucp4A flies. Com3

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C Figure 1. Overexpression of UCP4A rescues pink1 mutant phenotypes. A) Percentage of crushed thoraxes associated with WT flies, pink1-null mutants (pink1B9), and pink1-null mutants expressing UCP4A (pink1B9;Tub⬎ ucp4A: pink1B9;Tub-Gal4/UAS-ucp4A). At least 100 flies were scored for each genotype. B) Climbing abilities associated with flies of the indicated genotypes. At least 100 flies (3–5 d old) were scored for each genotype. C) Indirect flight muscles labeled with TUNEL (green) and DAPI (blue). D) TEM images of indirect flight muscles. Genotypes are indicated. Scale bars ⫽ 2 ␮m (top panels); 1 ␮m (bottom panels). E) Phase-contrast micrographs (top panels) and TEM images (bottom panels) of spermatids during the onion stage. TEM sections were obtained form flies that were 3–5 d old. In the top panels, black arrows indicate nuclei, and white arrows indicate nebenkerns. In the bottom panels, black arrows indicate mitochondrial derivatives, and white arrows indicate axonemes. Scale bars ⫽ 10 ␮m (top panels); 1 ␮m (bottom panels). Error bars ⫽ sd. **P ⬍ 0.01, ***P ⬍ 0.001; Student’s t test.

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ing force of ATP synthesis. As such, its reduction or loss indicates mitochondrial dysfunction and leads to mitophagy-associated degradation (39). We used a JC-10 assay to assess the mitochondrial membrane potential in different genetic backgrounds. The addition of CCCP resulted in the complete loss of membrane potential, which was reflected in a significant increase in the 525/590 nm fluorescence ratio (Fig. 2D). The fluorescence ratio was significantly higher in pink1


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Figure 2. Overexpression of UCP4A preserves mitochondrial function in pink1 mutant 3-d-old flies. A) Steady-state ATP levels for dissected thoraxes. Genotypes are indicated (nⱖ3). B) Rates of ATP synthesis for indicated genotypes. C) Rates of oxygen consumption associated with purified mitochondria. Genotypes are indicated, and pyruvate and malate were used as substrates (nⱖ4). D) Mitochondrial membrane potentials associated with indirect flight muscles. The JC-10 assay was used to assess membrane potentials. JC-10 fluorescence emissions were measured at 525 and 590 nm. The 525/590 ratio is presented to indicate the membrane potential. Error bars ⫽ sd. Significant differences were determined with the Student’s t test. ns, not significant. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

mutant cells than in WT cells, indicating that loss of pink1 results in the accumulation of depolarized mitochondria. In contrast, mild uncoupling via UCP4A expression increased the mitochondrial membrane potential of pink1 mutant cells (Fig. 2D). These results demonstrate that UCP4A-induced uncoupling protects pink1 mutant mitochondria from the loss of membrane potential, defective oxidative phosphorylation, and reduced mitochondrial ATP synthesis, thus maintaining mitochondrial integrity and function. UCP4A protects mitochondria from PD-related oxidative stress The mitochondrial ETC reaction is the primary source of cellular ROS, and ROS accumulation leads to oxidative stress and mitochondrial damage. Therefore, reductions in mitochondrial ROS may decrease oxidative damage and protect mitochondrial integrity. Indeed, decreasing ROS accumulation by antioxidants or the UCP4A PROTECTS AGAINST PARKINSON=S DISEASE

expression of superoxide dismutase protects animals from a range of stresses (40, 41). As UCP4A alleviated mitochondrial damage associated with the pink1 mutation, we reasoned that it would inhibit ROS production. We therefore examined the effect of increased mitochondrial uncoupling on ROS production by using DHE to measure cellular superoxide levels. Consistent with high levels of mitochondrial damage, pink1 mutant cells had 6-fold more superoxide than WT cells had. UCP4A overexpression significantly reduced the superoxide levels associated with pink1 mutant cells (Fig. 3A). We also measured the levels of hydrogen peroxide, another ROS, by H2DCFA staining. Consistent with the superoxide results, the high levels of hydrogen peroxide of pink1 mutant cells were largely reduced by the expression of UCP4A (Fig. 3B). As UCP4A expression in pink1 mutant cells reduced ROS production and preserved mitochondrial function, UCP4A may also increase cellular resistance to PD-related oxidative stresses. The ROS generators para5

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Figure 3. UCP4A expression protects against oxidative stress. A, B) Superoxide (A) and hydrogen peroxide (B) levels were measured with DHE and H2DCFA respectively, in the indicated genotypes. ROS levels are presented relative to WT, which was set to 1. C) Mitochondrial membrane potential of indirect flight muscles was measured with the JC-10 assay. Data for control, control ⫹ 20 mM paraquat, and Tub⬎ucp4A ⫹ 20 mM paraquat are shown. D, E) Survival curves for control (Da-Gal4/⫹) and UCP4A-overexpressing (Da⬎ucp4A: Da-Gal4/UAS-ucp4A) 3- to 5-d-old flies after exposure to 20 mM paraquat (D) or 5 mM rotenone (E). F, G) Expression of UCP4A extended the life span of both male (F) male and female (G) flies. Error bars ⫽ sd. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001, ****P ⬍ 0.0001; 2-way t test.

quat and rotenone cause oxidative damage and contribute to the pathogenesis of sporadic PD. We therefore asked whether UCP4A could protect animals from paraquat and rotenone. In WT flies, paraquat treatment lowered the mitochondrial membrane potential, and all the animals were dead after 96 h of exposure (Fig. 3C, D). Overexpression of UCP4A (Da⬎UCP4A) reduced the effects of paraquat, as these animals had a higher mitochondrial membrane potential and a longer life span (Fig. 3C, D). Treating animals with rote6

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none, which is a mitochondrial complex I inhibitor, results in ATP depletion, oxidative damage, and PD-like motor defects (42). Da⬎ucp4A flies survived longer than WT flies when exposed to rotenone (Fig. 3E). As UCP4A decreased cellular oxidative damage and protected cells under oxidative stress through a reduction in ROS production, we examined the effect of modulated mitochondrial uncoupling on the process of aging under normal conditions. UCP4A expression extended the life span of both male and female flies,


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indicating that protection of mitochondria against oxidative stress has significant positive effects on longevity (Fig. 3F, G). These results are consistent with a UCP4Ainduced decrease in ROS production and thus protection of the cells from oxidative stress. UCP4A suppresses parkin mutant phenotypes The loss of parkin causes mitochondrial degeneration within a subset of Drosophila tissues, and this results in male sterility and locomotor defects that phenocopy pink1 mutant phenotypes (12). Studies in model animals and cultured cells suggest that pink1 and parkin genetically interact and that overexpression of Parkin partially rescues the mitochondrial degeneration caused by the mutated pink1 (13, 14). However, it has

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also been suggested that Parkin has functions that are unrelated to PINK1 or that Parkin functions in one of the parallel pathways downstream of PINK1, as many pink1 suppressors fail to modify parkin phenotypes in Drosophila (33, 43– 47). Moreover, PINK1 is necessary to maintain mitochondrial function such as complex I activity, independent of recruitment of parkin for mitophagy (48). We therefore asked whether UCP4A affects parkin phenotypes in vivo. Loss-of-function parkin alleles exhibit phenotypes that include a crushed thorax, defective locomotion, and male sterility. All of these phenotypes were suppressed by UCP4A expression (Act-Gal4/ucp4A;park1: Act⬎ucp4A;park1; Fig. 4A, B). In addition, UCP4A expression abrogated the muscle degeneration associated

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Figure 4. UCP4A overexpression rescues parkin phenotypes. A) Percentage of crushed thoraxes associated with WT flies, parkin-null mutants (park: park1), and parkin-null mutants expressing UCP4A (Act⬎ucp4A;park1: Act-Gal4/UAS-ucp4A;park1). At least 100 flies were scored for each genotype. B) Climbing abilities associated with flies of the indicated genotypes. At least 100 flies (3–5 d old) were scored for each genotype. C) Mitochondrial membrane potential of indirect flight muscles was measured with the JC-10 assay. D) TUNEL (green) and DAPI (blue) staining of indirect flight muscles. E) TEM images of indirect flight muscles. Arrows indicate mitochondria. Sections were obtained from 3–5-d-old flies. Scale bar ⫽ 1 ␮m. Error bars ⫽ sd. **P ⬍ 0.01, ***P ⬍ 0.001; 2-way t test. UCP4A PROTECTS AGAINST PARKINSON=S DISEASE

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with parkin loss of function, as few TUNEL-positive cells were detected in the indirect flight muscles of the Act⬎ucp4A;park1 flies (Fig. 4D). TEM revealed that UCP4A expression also rescued mitochondrial impairment in the parkin mutant flies, as cristae structures were restored (Fig. 4E). Finally, UCP4A restored the mitochondrial membrane potential of the parkin flies (Fig. 4C). These results indicate that preserved mitochondria function likely contributed to the suppression of parkin phenotypes (Fig. 4C). Therefore, UCP4A protected against mitochondrial dysfunction and degeneration caused by parkin mutations.

whether ucp4A mutants were sensitive to paraquat and rotenone. When ucp4ADL animals were exposed to rotenone or paraquat, they survived for significantly less time than the controls (a precise ucp4AG1388 excision line) subjected to the same treatment (Fig. 5B, C). Moreover, ucp4A and pink1 double mutants exhibited significantly shorter life spans with paraquat or rotenone treatment compared with the life spans of the treated pink1 mutants (Fig. 5B, C). These results demonstrate that endogenous UCP4A helps to minimize the effects of oxidative stress.

Loss of UCP4A reduces resistance to oxidative stress

DISCUSSION

To obtain a ucp4A mutant, we mobilized the ucp4AG1388 P-element, which is inserted near the 5= untranslated region of ucp4A (Fig. 5A). One excision line (ucp4ADL) contained a 2.1 kb deletion, which removed nearly the entire ucp4A coding region (Fig. 5A). We confirmed this deletion with genomic sequencing. Thus, ucp4ADL represents a null allele of ucp4A. The ucp4ADL flies were viable and fertile and did not exhibit an obvious phenotype under normal conditions. We then asked

The mitochondrial membrane potential is critical for maintaining the ETC and for generating ATP. Mitochondrial uncoupling could result in the loss of mitochondrial membrane potential, which depletes the cell of energy and eventually leads to cell death. Mitochondrial uncoupling is associated with a variety of neurodegenerative conditions, depriving neurons of essential energy and making them more vulnerable to stressful conditions (32). A G2019S mutation in LRRK2 is a ucp4AG1388

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Figure 5. Loss of UCP4A increases sensitivity to oxidative stress. A) The ucp4A locus (top) and the ucp4ADL deletion line (bottom). An imprecise P-element excision was used to generate a 2.1 kb deletion in ucp4A. B, C) Paraquat-induced (B) and rotenone-induced (C) lethality of WT and pink1 mutant flies were enhanced by loss of UCP4A. Genotypes are indicated. Flies that were 3–5 d old were treated with 20 mM paraquat or 5 mM rotenone. At least 100 flies per genotype were assayed. Error bars ⫽ sd. *P ⬍ 0.05, ***P ⬍ 0.001; 2-way t test.

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common genetic cause of PD and is accompanied by increased levels of UCP2 and UCP4 and a subsequent loss of mitochondrial membrane potential (30). However, it has been proposed recently that the high expression of UCP2 detected in LRRK2-associated PD cells is elicited by elevated ROS levels and protects cells from ROS overload (49). Similarly, HtrA2 mutations reduce the mitochondrial membrane potential via mitochondrial uncoupling in a range of cell types, including neurons (31). Finally, pink1 and parkin mutant animals exhibit reductions in membrane potential, ETC efficiency, and bioenergetics, and these phenotypes most likely lead to mitochondrial degeneration. In the current study, however, expressing UCP4A in pink1 or parkin mutants did not further lower the mitochondrial membrane potential and ATP production; rather, it ameliorated these phenotypes. This process results from defective ATP synthesis rather than increased ATP consumption, as UCP4A expression not only rescued steady-state ATP levels but also rescued mitochondrial ATP respiration and synthesis. Therefore, the mitochondrial uncoupling described in PD models may be a secondary effect of mitochondrial dysfunction, and observed increases in UCPs may represent a natural defensive mechanism that protects against mitochondrial damage. ROS are generated in a wide range of normal physiological conditions, and in most tissues the main ROS source is the mitochondrial ETC. The accumulation of mitochondrial ROS induces oxidative damage of mitochondrial proteins, membranes, and DNA, which leads to mitochondrial dysfunction (40). Increased ROS levels and oxidative stress are common features of PD and are likely contributors to neuronal cell death (50). It has been proposed that mild uncoupling via the induction of UCPs causes a portion of the mitochondrial proton gradient to leak from the intermembrane space into the matrix, thereby reducing both ROS and oxidative damage (26, 27, 51, 52). It has also been suggested that the PINK1/Parkin pathway promotes neuronal survival by reducing levels of oxidative stress. In support of this hypothesis, neurodegeneration associated with PINK1 inactivation is suppressed by antioxidants, including superoxide dismutase 1 and vitamin E. In addition, glutathione S-transferase S1 prevents the degeneration of DA neurons in parkin mutants (53, 54). Mammalian UCP4 has been shown to mediate a shift in energy metabolism and to reduce ROS production, thus increasing the resistance to oxidative and mitochondrial stress (55, 56). Much as with mammalian UCP4 and UCP2, we found that fly UCP4A protected against paraquat and rotenone by stabilizing the mitochondrial membrane potential and reducing the formation of ROS under oxidative stress. In pink1 or parkin mutants, the PINK1/Parkin pathway was disrupted, resulting in the accumulation of ROS and dysfunctional mitochondria. In this context, UCP4A expression induced proton leakage across the inner mitochondrial membrane, thereby dissipating the hyperpolarized membrane potential and minimizing ROS production UCP4A PROTECTS AGAINST PARKINSON=S DISEASE

during oxidative phosphorylation. These effects protected pink1 and parkin mutant tissues from the pathologic effects of PD. Moreover, genetic deletion of ucp4A resulted in sensitivity to oxidative stress, supporting the hypothesis that UCP4A and associated mitochondrial uncoupling promotes cell viability in stress conditions. Despite encouraging results in animal models of PD, antioxidant compounds have failed to slow disease progression in humans (50). Using UCPs to induce mild uncoupling may provide an alternate means of reducing oxidative stress by inhibiting ROS formation rather than degrading them and thereby may provide cell protection in PD. Age is the main risk factor for PD and other neurodegenerative diseases. In PD, environment toxins and genetic mutations induce mitochondrial damage, which reduces the efficiency of the ETC and promotes electron leakage and the accumulation of ROS (57). Mitochondrial damage accumulates during the aging process, eventually reaching levels where endogenous defense mechanisms are no longer effective. Loss of PINK1 or Parkin blocks the degradation of damaged mitochondria, leading to their accumulation. This effect can be quite remarkable in cells with high energy demands, such as muscles and spermatids in flies (13, 14). Efforts to correct dysfunctional mitochondria in PD have been ineffective, probably because there are many causes of mitochondria dysfunction. A more effective therapeutic approach may be needed to protect mitochondria against insults relevant to the pathogenesis of PD, thereby preventing mitochondrial damage rather than attempting to treat it (5). Indeed, expression of UCP4A in some PD models ameliorates the phenotypes associated with mitochondrial membrane potential, ROS accumulation, ETC efficiency, and ATP synthesis. In this way, UCP4A maintains mitochondrial integrity and profoundly affects the pathologic course of PDs. Moreover, there is evidence that mitochondrial dysfunction causes aging (58). One way of increasing life span is to activate beneficial stress-defense pathways by applying a moderate level of stress (59). Indeed, mild mitochondrial distress preserves mitochondrial function and prolongs life span in several organisms (60 – 62). We observed that flies expressing UCP4A lived longer than WT flies in normal conditions, further supporting the notion that protecting mitochondria during the aging process increases longevity. Although both PINK1 and Parkin function in the mitophagy pathway, the evidence suggests that PINK1 and Parkin also have independent functions and contribute to PD via mechanisms that do not involve mitophagy. In Drosophila, several factors, including VCP, PGAM5, Ret, HtrA2, and Acon, modulate pink1 phenotypes but fail to modify parkin phenotypes, suggesting, for example, that PINK1 has additional downstream targets (33, 43– 47). Nevertheless, phenotypes of pink1 and parkin mutant animals are rescued by regulating mitochondrial homeostasis from increasing mitofission or decreasing mitofusion (16, 63, 64). In the current study, the direct regulation of mitochon9

drial activity by UCP4A rescued male sterility, muscle degeneration, and locomotion defects of pink1 and parkin mutants. These results suggest that although Parkin and PINK1 may have independent functions, they both contribute to PD pathogenesis by affecting mitochondrial quality control. It is unlikely that UCP4A acts directly on the PINK1/Parkin pathway; rather, it represents an upstream protective pathway that regulates cellular integrity. The authors thank Y. Wang and Drs. J. Fang and Q. Bai for technical support and valuable comments. The authors thank the Bloomington Stock Center (Indiana University, Bloomington, IN, USA), Dr. Stephen Helfand (Brown University, Providence, RI, USA), Dr. Jongkyeong Chung (Seoul National University, Seoul, Korea), Dr. Leo J. Pallanck (University of Washington, Seattle, WA, USA), and Dr. Bingwei Lu (Stanford University School of Medicine, Stanford, CA, USA) for fly stocks. This work was supported by a 973 grant (2011CB812702) from the Chinese Ministry of Science and Technology (to T.W). The authors declare no conflicts of interest.

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