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Chicken DT40 cell line lacking DJ-1, the gene responsible for familial Parkinson's disease, displays mitochondrial dysfunction
Neuroscience Research 77 (2013) 228–233
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Chicken DT40 cell line lacking DJ-1, the gene responsible for familial Parkinson’s disease, displays mitochondrial dysfunction Eiko N. Minakawa a , Hodaka Yamakado a , Atsushi Tanaka b,1 , Kengo Uemura a , Shunichi Takeda c , Ryosuke Takahashi a,∗ a
Department of Neurology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, MSC 3704, Bethesda, MD 20892-3704, USA c Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan b
a r t i c l e
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Article history: Received 28 June 2013 Received in revised form 8 September 2013 Accepted 13 September 2013 Available online 21 September 2013 Keywords: Parkinson’s disease DJ-1 DT40 Mitochondria Oxidative stress
a b s t r a c t Parkinson’s disease (PD) is the most common neurodegenerative movement disorder mainly due to gradual loss of dopaminergic neurons in the substantia nigra. Although the causative genes for autosomal recessive PD, Parkin, PINK1 and DJ-1, share a common pathway, at least in part, in mitochondrial quality control and protein quality control, their precise relationship remains elusive. Previous studies suggested the limitation of gene-modified mice model to solve this problem. DT40 is an avian leukosis virus-induced chicken B cell line with an exceptionally high ratio of targeted to random DNA integration, which enables efficient targeted disruption of multiple genes of interest. We generated DJ-1-deficient DT40 cells and analyzed PD-related phenotypes. These cells exhibited vulnerability to oxidative stress, mitochondrial dysfunction and fragmentation. Importantly, we showed that mitochondrial membrane potential and morphology are available for the phenotype analysis in DT40. These results suggest that genetically engineered DT40 cells would serve as a relevant model of PD, and help understand the genetic and functional relationship among multiple causative genes. Furthermore, in line with the recent concept of PD as a systemic disorder, elucidating the pathomechanism of PD using DT40 would lead to the development of noninvasive diagnostic tools and drug screening assays using patient-derived lymphocytes. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction Parkinson’s disease (PD) is the most common neurodegenerative movement disorder that causes progressive motor symptoms mainly due to gradual loss of dopaminergic neurons in the substantia nigra pars compacta (de Lau and Breteler, 2006). Treatments that provide neuroprotection and/or disease-modifying
Abbreviations: PD, Parkinson’s disease; ROS, reactive oxygen species; H2 O2 , hydrogen peroxide; carboxy-H2 DCFDA, 5-(and-6)-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate; AV, Annexin-V; PI, propium iodide; TMRE, tetramethylrhodamine ethyl ester; CCCP, carbonyl cyanide m-chlorophenylhydrazone; MMP, mitochondrial membrane potential. ∗ Corresponding author. E-mail addresses: [email protected] (E.N. Minakawa), [email protected] (H. Yamakado), [email protected] (A. Tanaka), [email protected] (K. Uemura), [email protected] (S. Takeda), [email protected] (R. Takahashi). 1 Present address: Central Laboratory for Research and Education, Faculty of Medicine, Yamagata University, 2-2-2 Iida-nishi, Yamagata-shi, Yamagata 9909585, Japan.
effects remain an unmet clinical need because currently available dopamine replacement therapies only partially improve the symptoms (Meissner et al., 2011). Though PD is typically sporadic in origin, identification of genes responsible for familial PD has provided clues to understand the pathogenesis of the more common, sporadic form of PD (Moore and Dawson, 2008). Parkin, PINK1 and DJ-1 are causative genes of autosomal recessive PD, in which clinical phenotype is often indistinguishable from early-onset idiopathic PD (Kitada et al., 1998; Valente et al., 2004; Bonifati et al., 2003). Identification of these genes has promoted generation of gene-modified models to investigate the pathomechanisms of the disease (Shulman et al., 2011). Recent studies have revealed that these genes share, at least in part, a common pathway in mitochondria quality control and protein quality control. Genetic studies using Parkin and PINK1 knockout Drosophila indicated that PINK1 acts upstream of Parkin (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Further studies elucidated that Parkin and PINK1 share a common mitochondrial quality control pathway conserved in mammalian cells (Narendra and Youle, 2011; Matsui et al., 2013). Meanwhile, it is still controversial whether DJ-1 works in association with PINK1/Parkin
0168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. http://dx.doi.org/10.1016/j.neures.2013.09.006
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pathway (Yang et al., 2006; Exner et al., 2007; Hao et al., 2010; Thomas et al., 2011). Although several previous studies suggested that Parkin and/or PINK1 interact with DJ-1 to promote protein degradation (Olzmann et al., 2007; Xiong et al., 2009), the precise genetic and functional relationships between these genes remain elusive. Genetic mammalian models of autosomal recessive PD, such as Parkin-, PINK1- and DJ-1-knockout mice, have failed to recapitulate the hallmark features of PD, especially the progressive loss of nigral dopaminergic neurons (Moore and Dawson, 2008). Furthermore, aged triple knockout mice lacking Parkin, DJ-1, and PINK1 do not exhibit PD-related phenotypes (Kitada et al., 2009). These observations raise the possibility of functional redundancy among mice genes and suggest the limitation of gene-modified mice model to clarify the relationship among these genes (Shulman et al., 2011). A novel model using higher vertebrates would be helpful to further reveal their functional association. Here we established DJ-1−/− DT40 cells and examined their phenotype to evaluate whether DT40 cells could be used as a relevant model of PD. DT40 is an avian leukosis virus-induced chicken B cell line with an exceptionally high ratio of targeted to random DNA integration of a transfected genomic DNA fragment to its homologous genomic locus (Buerstedde and Takeda, 1991). This feature enables efficient targeted gene disruption in vertebrate cells without generating a knockout animal model. We report that DJ-1−/− DT40 cells recapitulate multiple phenotypes compatible to those of DJ-1 deficient mammalian cells and that mitochondrial membrane potential and morphology are available as a readout of phenotype analysis in DT40 cells.
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Laboratories) at 550 V and 25 F. Drug-resistant clones were selected with medium containing 0.5 g/ml puromycin (Nacalai Tesque) or 1 mg/ml histidinol (Nacalai Tesque). 2.3. Genomic Southern blotting Genomic DNAs of the wild type and candidate clones of DJ-1+/− or DJ-1−/− DT40 cells were extracted and digested with BglII for genomic Southern blotting analysis. To generate a probe for screening gene target events, 0.5-kb DNA fragment generated by PCR of genomic DNA using the primers 5 -AGAGATGCCAACTGGAAGCA-3 and 5 GTTTGTGAGCAGAGTGACAT-3 was cloned into TOPO vector (Life Technologies). Then the 430 bp digestion product of the cloned plasmid with AflII was used as the probe. Southern blotting was performed using standard procedures. 2.4. RT-PCR To confirm the gene disruption by RT-PCR, total RNA from wild type or DJ-1−/− cells was extracted using Sepasol-RNA I Super (Nacalai Tesque). First-strand cDNA synthesis was performed using SuperScript III First-Strand Synthesis System for RT-PCR (Life Technologies). All procedures were performed according to manufacturer’s protocols. The subsequent PCR was performed using primers 5 -ATCAAGGTGACTGTTGCAGG-3 and 5 GTGCCTTCACTTGTTCAGCC-3 , which hybridize sequences in exon 3 and 7 respectively. 2.5. Western blotting
2. Materials and methods 2.1. Cell culture DT40 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% chicken serum, 100 units/ml penicillin and 100 g/ml streptomycin at 39 ◦ C with 5% CO2 . 2.2. Generation of DJ-1−/− DT40 Cells DJ-1 gene disruption constructs (DJ-1-puro, DJ-1-his) were generated from genomic PCR products combined with puromycinor histidinol-resistant selection cassettes flanked by loxP sites using MultiSite Gateway technology (Invitrogen). All procedures were performed according to the manufacturer’s instructions. The drug resistant cassettes were inserted in exon 4 of DJ-1 gene. Genomic DNA sequences of wild type DT40 cells were amplified using primers 5 -GGGGACAACTTTGTATAGAAAAGTTGATGGCCTCGAAAAGAGCGTTGGTGATTCTG-3 and 5 -GGGGACTGCTTTTTTGTACAAACTTGCCAGGCAGGACAATGACATC-3 for the 5 -arm and 5 -GGGGACAGCTTTCTTGTACAAAGTGGAGGTAACCTTGGAGCTCAGA-3 and 5 -GGGGACAACTTTGTATAATAAAGTTGGCAATCCCTGTGTAAGGCAG-3 for the 3 -arm. To generate the 5 - and 3 -arm entry clones, 1.5 kb from the 5 - and 4.4 kb from the 3 -arm were subcloned by BP recombination into the donor vectors pDONR P4-P1R and pDONR P2R-P3, respectively. To generate the targeting vector by LR recombination, we used the 5 -arm entry clone, 3 -arm entry clone, pDEST DTA-MLS, and Puro or His entry clones (Iiizumi et al., 2006). To generate DJ-1+/− cells, DJ-1-puro construct was linearized by AscI and transfected into wild type DT40 cells. To generate DJ-1−/− cells, DJ-1-his construct was linearized using AscI and transfected to DJ-1+/− cells. DNA transfection and selection were performed as described previously (Kohzaki et al., 2010). Briefly, 10–30 g of linearized targeting vectors was electroporated into 107 DT40 cells with Gene Pulser apparatus (Bio-Rad
To confirm the absence of DJ-1 protein in DJ-1−/− cells by Western blotting, cells were lysed on ice with 2× lysis buffer (0.125 M Tris–HCl, 4% SDS, 10% -mercaptoethanol, 0.02% BPB, 20% glycerol) and boiled for 15 min. SDS-PAGE and immunoblotting were carried out using standard procedures. Rabbit polyclonal anti-DJ-1 antibody was obtained from Novus. Rabbit polyclonal anti-GAPDH antibody was obtained from Sigma. 2.6. Measurement of intracellular reactive oxygen species (ROS) accumulation Wild type or DJ-1−/− DT40 cells were treated with 10 M hydrogen peroxide (H2 O2 ) for 24 h and monitored for intracellular ROS accumulation by labeling with 5-(and-6)-carboxy2 ,7 -dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA, Life Technologies) according to the manufacturer’s protocol with slight modification. Briefly, cells were incubated in 5 M carboxyH2 DCFDA in PBS with 1 mM CaCl2 and 0.5 mM MgCl2 for 30 min, followed by being washed once with ice-cold PBS and resuspended in 2 ml of PBS. The fluorescence intensity was measured using a flow cytometer (FACSCalibur, Becton Dickinson). 2.7. Analysis of hydrogen-peroxide-induced cell death Wild type or DJ-1−/− DT40 cells were treated with 10 M H2 O2 for 24 h and analyzed for apoptotic marker and plasma membrane integrity with Annexin-V and propidium iodide staining according to the manufacturer’s protocol (Roche Applied Science). Briefly, cells were washed once with PBS and incubated with labeling solution containing both Annexin-V and propidium iodide for 15 min at 25 ◦ C. Fluorescence intensity was measured using a flow cytometer (FACSCalibur, Becton Dickinson). The results were analyzed using CellQuest software. Gating was determined empirically in one experiment and applied to all subsequent experiments.
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2.8. Measurement of mitochondria membrane potential Mitochondria membrane potential was analyzed using MitoTracker Green FM, MitoTracker Red CMXRos (Life Technologies) and tetramethylrhodamine ethyl ester (TMRE, Life Technologies). Cells were stained for 30 min with 50 nM MitoTracker Red CMXRos, replated onto a poly-d-lysine coated chambered coverglass (Nunc) and imaged using an inverted confocal microscope (LSM510 Meta, Zeiss). As a positive control for mitochondria membrane potential loss, DT40 cells were incubated with 10 M of carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 60 min, stained with MitoTracker Red CMXRos and imaged as described above. Flow cytometric analysis was performed using cells stained with 100 nM TMRE or 25 nM MitoTracker Green FM for 30 min. Analysis was performed using FACSCalibur (Becton Dickinson) and CellQuest software. At least 10,000 cells were analyzed for the corresponding fluorescence in each experiment. Gating was determined empirically in one experiment and applied to all subsequent experiments. 2.9. Analysis of mitochondrial morphology by immunostaining DT40 cells were attached onto a poly-d-lysine coated coverslip for 1 h at 39 ◦ C with 5% CO2 . The coverslips were gently immersed into 4% paraformaldehyde in PBS for 15 min for fixation and then into 0.1% NP-40 in PBS for 5 min for permeabilization. After blocking with 1% BSA in PBS for 30 min, the samples were incubated with polyclonal anti-Tom20 antibody (SantaCruz) in 1% BSA/PBS at 4 ◦ C overnight. The samples were then stained with Alexa Fluor 568 (Life Technologies) in 1% BSA/PBS for one hour at 25 ◦ C and mounted with Vectashield mounting media with DAPI (Vector Laboratories). Images were randomly obtained using an inverted confocal microscope (LSM510 Meta; Zeiss) and analyzed by Spot Detector Version 3.0 BioApplication (Thermo Fisher Scientific). The size of each spot surrounded by Tom20-positive signal was analyzed. The number of the spots per cell was also counted. The average size of spot was determined for 113 cells in wild type and 114 cells in DJ-1−/− cells.
has been suggested to have multiple possible functions, a consistent finding is that DJ-1 protects against oxidative stress in vitro and in vivo (Cookson, 2010). Therefore, we first investigated whether this was recapitulated in DT40 cells. We treated cells with H2 O2 and determined intracellular accumulation of ROS by flow cytometric analysis with 5-(and-6)-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA). DJ-1−/− DT40 cells showed significantly increased accumulation of intracellular ROS compared to wild type cells (Fig. 2A). To examine vulnerability of DJ-1−/− DT40 cells to oxidative stress, we analyzed H2 O2 -induced cell death. At 24 h after H2 O2 treatment, we measured the ratio of viable cells, early-apoptotic cells, and late-apoptotic and necrotic cells by flow cytometric analysis. We found that the ratio of viable cells was significantly lower and that early-apoptotic and late-apoptotic and necrotic cells were significantly higher in DJ-1−/− cells than in wild type cells (Fig. 2B and C). These results were compatible with the notion that DJ-1 has physiological antioxidant property in DT40 cells. 3.3. Decreased mitochondrial membrane potential in DJ-1−/− DT40 cells Mitochondrial dysfunction has long been implicated in the etiopathogenesis of PD (Imai and Lu, 2011). Indeed, DJ-1-deficient mammalian cells show mitochondrial dysfunction (Krebiehl et al., 2010). To analyze whether loss of DJ-1 resulted in mitochondrial dysfunction in DT40 cells, we evaluated mitochondrial membrane potential (MMP) using the following two MMP-dependent dyes, MitoTracker Red CMXRos and tetramethylrhodamine ethyl ester
2.10. Statistical analysis In phenotype analyses, experiments were repeated independently three times using one representative DJ-1−/− clone after having confirmed by a preliminary experiment that two or three different DJ-1−/− clones exhibit the same phenotype. GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA) was used for all statistical analyses. Statistical tests are described in the figure legends. 3. Results 3.1. Generation of DJ-1−/− DT40 Cells We disrupted two DJ-1 alleles by sequentially transfecting the two targeting constructs (DJ-1-puro, DJ-1-his) carrying puromycin or histidinol resistance gene into DT40 cells (Fig. 1A). Disruption of both alleles was confirmed by genomic Southern blotting (Fig. 1B). Absence of detectable mRNA or protein was confirmed by RT-PCR using primers designed to flank drug-resistance gene (Fig. 1C) and western blotting (Fig. 1D). 3.2. Increased vulnerability to oxidative stress in DJ-1−/− DT40 cells DJ-1 was originally identified as an oncogene and was found to be the causative gene of autosomal recessive Parkinson’s disease, PARK7 (Nagakubo et al., 1997; Bonifati et al., 2003). Although DJ-1
Fig. 1. Targeted disruption of DJ-1 in DT40 cells. (A) Schematic representation of the chicken DJ-1 locus and the gene-targeting constructs. Black squares indicate exon 2–7. The restriction enzyme sites and the probe used for genomic Southern blot analysis are shown. (B) Southern blot analysis of BglII-digested genomic DNA from wild type, DJ-1+/−, and DJ-1−/− DT40 cells. (C) Representative RT-PCR analysis of wild type and DJ-1−/− cells. The forward primer was designed in exon 3, and the reverse primer was designed in exon 7. The expected length of amplicon from wild-type DT40 cells is 460 bp. No band corresponding to DJ-1 was observed in DJ-1 knockout cells. (D) Western blot analysis of whole cell lysate from wild type and DJ-1−/− DT40 cells. Blots were probed with anti-DJ-1 polyclonal antibody and anti-GAPDH polyclonal antibody. No band corresponding to DJ-1 was observed.
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Fig. 2. Increased vulnerability to oxidative stress in DJ-1 −/− DT40 cells. (A) Flow cytometric analysis of intracellular ROS. Wild type or DJ-1 −/− DT40 cells were treated with 10 M H2 O2 for 24 h and stained with carboxy-H2 DCFDA, a ROS indicator. The fluorescence intensity was measured by flow cytometry. The amount of intracellular ROS after H2 O2 treatment was significantly greater in DJ-1 −/− cells. Error bars indicate mean ± SEM values from three independent experiments. *P < 0.05, N.S.; not significant by Bonferroni multiple comparison test. (B) Representative flow cytometric analysis of cell viability after H2 O2 treatment. Wild type or DJ-1 −/− DT40 cells were treated with 10 M H2 O2 for 24 hours and stained with Annexin-V (AV) and propium iodide (PI). Lower right quadrant (AV positive and PI negative) corresponds to early-apoptotic cells, and upper right quadrant (AV and PI doublepositive) corresponds to late-apoptotic and necrotic cells. Cells not stained with AV and PI (lower left quadrant) are viable cells. (C) Quantification of viable, early-apoptotic, and late-apoptotic and necrotic cells after treatment with 10 M H2 O2 for 24 h. The percentage of the cells in lower left (LL), lower right (LR) or upper right (UR) quadrant shown in (B) were quantified and were analyzed by Bonferroni multiple comparison test. Error bars indicate mean ± SEM values from three independent experiments. *P < 0.05, N.S.; not significant.
(TMRE). The staining with the former reagent showed a decrease of MMP in DJ-1−/− DT40 cells, as indicated by lower levels of staining in the mutant cells compared to wild type DT40 cells (Fig. 3A). We confirmed this result by detecting the intensity of TMRE staining, a quantitative indicator of membrane potential, by flow cytometry. The MMP of DJ-1−/− DT40 cells was significantly decreased compared to wild type cells (Fig. 3B and C). To exclude the possibility that the decreased staining with these dyes was caused by a decrease in the number of mitochondria, we stained the cells with MitoTracker Green, which is a MMP-independent dye. The intensity of MitoTracker Green signals did not differ between wild type and DJ-1−/− cells. This indicated that the reduced staining with MitoTracker Red CMXRos or TMRE was not due to decrease in the number of mitochondria in DJ-1−/− cells (Fig. 3D). Overall, these results suggested that loss of DJ-1 in DT40 cells resulted in decreased mitochondrial membrane potential. 3.4. Fragmented mitochondria in DJ-1−/− DT40 cells Recent studies have consistently demonstrated that DJ-1deficient mammalian cells exhibit mitochondrial fragmentation (Krebiehl et al., 2010; Irrcher et al., 2010). To confirm the reproducibility of this phenotype in DJ-1−/− DT40 cells, we immunostained mitochondria with Tom20, a mitochondrial outer membrane specific protein, and imaged by laser confocal microscope (Fig. 4A). We determined the average size of mitochondria using Spot Detector software by analyzing the size of each spot surrounded by Tom20-positive signal and the number of the spots per cell. Remarkably, in DJ-1−/− DT40 cells, the average size of these spots was significantly decreased (Fig. 4B) and the number of these spots per cell was significantly increased (Fig. 4C) compared to wild type DT40 cells. 4. Discussion In the present study, we generated DJ-1 deficient DT40 cells, which exhibited vulnerability to oxidative stress, mitochondrial dysfunction and mitochondrial fragmentation. We utilized DT40, a lymphocyte cell line, as a novel PD cellular model. The rationale for our approach is that Parkin, PINK1 and DJ-1 are highly conserved across species and are expressed in lymphocytes. Moreover, analysis of lymphocytes would contribute to
understanding the pathomechanism of PD. This is evidenced by subclinical PD-related phenotypes in lymphocytes or lymphoblasts of PD patients. Mitochondrial dysfunction and increased oxidative stress were observed in lymphocytes of PD patients (Müftüoglu et al., 2004; Prigione et al., 2009). Lymphoblasts established from a PD patient with DJ-1 mutation exhibited abnormal mitochondrial fragmentation (Irrcher et al., 2010). These subclinical phenotypes are consistent with the phenotype of DJ-1-deficient DT40 cells. These lines of evidence support the recent concept of PD as a systemic disorder (Imai and Lu, 2011) and demonstrates the validity of our approach. Although the effect of DJ-1 loss in DT40 cells on ROS accumulation and mitochondrial membrane potential were relatively small compared to previous studies, we speculate that this was because of the highly rapid turnover of DT40 cells. Furthermore, although the mitochondrial morphology is relatively fragmented in wild type DT40 cells compared to those typically observed in adherent culture cell lines, the difference in the quantitated Tom20-positive area indicated that loss of DJ-1 led to further mitochondrial fragmentation in DT40 cells. The advantage of this approach compared to the currentlyavailable cellular models including induced pluripotent stem cells-derived lines from patients, mouse embryonic fibroblasts from knockout mice and RNAi approaches, are the efficacy and consistency in generating and analyzing the effect of multiple gene disruption. These advantages rely on the following multiple unique characteristics of this cell line. Firstly, DT40 cell line exhibits the extraordinary high ratio of targeted to random DNA integration (Buerstedde and Takeda, 1991). This ensures highly efficient multiple targeted gene disruption in a single cell using seven different selection markers, which can be recycled by the cre/loxP (Winding and Berchtold, 2001; Arakawa et al., 2001). Secondly, although RNAi technology has been widely used in mammalian cell lines for targeted knockdown, the residual mRNA is always problematic for interpretation of the resulting phenotype (Neumann et al., 2010). Genetically engineered knockout of DT40 helps overcome this problem. The fact that wild type and gene-disrupted DT40 cells have completely identical genetic background also contributes to increase consistency in phenotype analysis. Moreover, DT40 cells have a short doubling time (∼8 h) and are genetically stable even after repeated passages compared to mammalian cells including mouse embryonic fibroblasts. These also help increase efficiency and consistency in phenotype analysis.
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Fig. 4. Fragmented mitochondria in DJ-1−/− DT40 cells. (A) Representative imaging of wild type and DJ-1−/− DT40 cells immunostained with anti-Tom20 antibody. Mitochondria were fragmented in DJ-1−/− cells. (B) Quantification of mitochondrial size. Randomly obtained images from anti-Tom20-stained DT40 cells were analyzed by Spot Detector Version 3.0 BioApplication. The average size of Tom20-positive spots was significantly smaller in DJ-1−/− cells. Error bars indicate mean ± SEM values. *P < 0.05 by unpaired t-test. (C) Quantification of mitochondrial number. The number of Tom20-positive spots per cell was significantly larger in DJ-1−/− cells. Error bars indicate mean ± SEM values. *P < 0.05 by unpaired t-test.
Fig. 3. Decreased mitochondrial membrane potential in DJ-1−/− DT40 cells. (A) Representative imaging of wild type and DJ-1−/− DT40 cells stained with MitoTracker Red CMXRos. DJ-1−/− cells had lower staining intensity of mitochondria than wild-type cells. CCCP treatment was performed as a positive control. (B) Representative flow cytometric analysis of TMRE staining intensity. (C) Quantification of TMRE staining. DJ-1−/− cells had significantly lower TMRE staining. Three independent experiments were performed and analyzed by unpaired t-test. Error bars indicate mean ± SEM values. *P < 0.01. (D) Quantification of MitoTracker GreenFM staining from flow cytometric analysis. Three independent experiments were performed and analyzed by unpaired t-test. Error bars indicate mean ± SEM values. N.S., not significant.
All these features make DT40 cell line attractive for analyzing effects of gene disruption as well as functional relationships among multiple genes. These advantages of DT40 cell lines have been previously taken to analyze the functions of B cell antigen receptor signaling, histone gene function, RNA processing, DNA repair, cell cycle, calcium signaling and autophagy (Winding and Berchtold, 2001; Alers et al., 2011). As accumulating evidence suggests the primary role of mitochondrial dysfunction in the pathomechanism of PD (Schapira, 2012), it is crucial to evaluate mitochondria function in PD models. We showed that mitochondrial membrane potential and morphology are available as a readout of phenotypic analysis in DT40 cells. Generation and functional analyses of double- or tripleknockout DT40 cell model of Parkin, PINK1 and DJ-1 will help further reveal the functional association among these genes in the maintenance of mitochondria as well as in the pathophysiology of PD. Furthermore, elucidating the pathomechanism of PD using DT40 lymphocyte line could lead to the development of novel tools to utilize patients’ lymphocytes as noninvasive diagnostic or therapeutic biomarkers. This approach also has a great potential for developing high-throughput assays for drug screening using patient-derived lymphocytes. Conflict of interest The authors have no conflict of interest to declare.
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Chicken DT40 cell line lacking DJ-1, the gene responsible for familial Parkinson’s disease, displays mitochondrial dysfunction Eiko N. Minakawa a , Hodaka Yamakado a , Atsushi Tanaka b,1 , Kengo Uemura a , Shunichi Takeda c , Ryosuke Takahashi a,∗ a
Department of Neurology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, MSC 3704, Bethesda, MD 20892-3704, USA c Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan b
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 8 September 2013 Accepted 13 September 2013 Available online 21 September 2013 Keywords: Parkinson’s disease DJ-1 DT40 Mitochondria Oxidative stress
a b s t r a c t Parkinson’s disease (PD) is the most common neurodegenerative movement disorder mainly due to gradual loss of dopaminergic neurons in the substantia nigra. Although the causative genes for autosomal recessive PD, Parkin, PINK1 and DJ-1, share a common pathway, at least in part, in mitochondrial quality control and protein quality control, their precise relationship remains elusive. Previous studies suggested the limitation of gene-modified mice model to solve this problem. DT40 is an avian leukosis virus-induced chicken B cell line with an exceptionally high ratio of targeted to random DNA integration, which enables efficient targeted disruption of multiple genes of interest. We generated DJ-1-deficient DT40 cells and analyzed PD-related phenotypes. These cells exhibited vulnerability to oxidative stress, mitochondrial dysfunction and fragmentation. Importantly, we showed that mitochondrial membrane potential and morphology are available for the phenotype analysis in DT40. These results suggest that genetically engineered DT40 cells would serve as a relevant model of PD, and help understand the genetic and functional relationship among multiple causative genes. Furthermore, in line with the recent concept of PD as a systemic disorder, elucidating the pathomechanism of PD using DT40 would lead to the development of noninvasive diagnostic tools and drug screening assays using patient-derived lymphocytes. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction Parkinson’s disease (PD) is the most common neurodegenerative movement disorder that causes progressive motor symptoms mainly due to gradual loss of dopaminergic neurons in the substantia nigra pars compacta (de Lau and Breteler, 2006). Treatments that provide neuroprotection and/or disease-modifying
Abbreviations: PD, Parkinson’s disease; ROS, reactive oxygen species; H2 O2 , hydrogen peroxide; carboxy-H2 DCFDA, 5-(and-6)-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate; AV, Annexin-V; PI, propium iodide; TMRE, tetramethylrhodamine ethyl ester; CCCP, carbonyl cyanide m-chlorophenylhydrazone; MMP, mitochondrial membrane potential. ∗ Corresponding author. E-mail addresses: [email protected] (E.N. Minakawa), [email protected] (H. Yamakado), [email protected] (A. Tanaka), [email protected] (K. Uemura), [email protected] (S. Takeda), [email protected] (R. Takahashi). 1 Present address: Central Laboratory for Research and Education, Faculty of Medicine, Yamagata University, 2-2-2 Iida-nishi, Yamagata-shi, Yamagata 9909585, Japan.
effects remain an unmet clinical need because currently available dopamine replacement therapies only partially improve the symptoms (Meissner et al., 2011). Though PD is typically sporadic in origin, identification of genes responsible for familial PD has provided clues to understand the pathogenesis of the more common, sporadic form of PD (Moore and Dawson, 2008). Parkin, PINK1 and DJ-1 are causative genes of autosomal recessive PD, in which clinical phenotype is often indistinguishable from early-onset idiopathic PD (Kitada et al., 1998; Valente et al., 2004; Bonifati et al., 2003). Identification of these genes has promoted generation of gene-modified models to investigate the pathomechanisms of the disease (Shulman et al., 2011). Recent studies have revealed that these genes share, at least in part, a common pathway in mitochondria quality control and protein quality control. Genetic studies using Parkin and PINK1 knockout Drosophila indicated that PINK1 acts upstream of Parkin (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Further studies elucidated that Parkin and PINK1 share a common mitochondrial quality control pathway conserved in mammalian cells (Narendra and Youle, 2011; Matsui et al., 2013). Meanwhile, it is still controversial whether DJ-1 works in association with PINK1/Parkin
0168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. http://dx.doi.org/10.1016/j.neures.2013.09.006
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pathway (Yang et al., 2006; Exner et al., 2007; Hao et al., 2010; Thomas et al., 2011). Although several previous studies suggested that Parkin and/or PINK1 interact with DJ-1 to promote protein degradation (Olzmann et al., 2007; Xiong et al., 2009), the precise genetic and functional relationships between these genes remain elusive. Genetic mammalian models of autosomal recessive PD, such as Parkin-, PINK1- and DJ-1-knockout mice, have failed to recapitulate the hallmark features of PD, especially the progressive loss of nigral dopaminergic neurons (Moore and Dawson, 2008). Furthermore, aged triple knockout mice lacking Parkin, DJ-1, and PINK1 do not exhibit PD-related phenotypes (Kitada et al., 2009). These observations raise the possibility of functional redundancy among mice genes and suggest the limitation of gene-modified mice model to clarify the relationship among these genes (Shulman et al., 2011). A novel model using higher vertebrates would be helpful to further reveal their functional association. Here we established DJ-1−/− DT40 cells and examined their phenotype to evaluate whether DT40 cells could be used as a relevant model of PD. DT40 is an avian leukosis virus-induced chicken B cell line with an exceptionally high ratio of targeted to random DNA integration of a transfected genomic DNA fragment to its homologous genomic locus (Buerstedde and Takeda, 1991). This feature enables efficient targeted gene disruption in vertebrate cells without generating a knockout animal model. We report that DJ-1−/− DT40 cells recapitulate multiple phenotypes compatible to those of DJ-1 deficient mammalian cells and that mitochondrial membrane potential and morphology are available as a readout of phenotype analysis in DT40 cells.
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Laboratories) at 550 V and 25 F. Drug-resistant clones were selected with medium containing 0.5 g/ml puromycin (Nacalai Tesque) or 1 mg/ml histidinol (Nacalai Tesque). 2.3. Genomic Southern blotting Genomic DNAs of the wild type and candidate clones of DJ-1+/− or DJ-1−/− DT40 cells were extracted and digested with BglII for genomic Southern blotting analysis. To generate a probe for screening gene target events, 0.5-kb DNA fragment generated by PCR of genomic DNA using the primers 5 -AGAGATGCCAACTGGAAGCA-3 and 5 GTTTGTGAGCAGAGTGACAT-3 was cloned into TOPO vector (Life Technologies). Then the 430 bp digestion product of the cloned plasmid with AflII was used as the probe. Southern blotting was performed using standard procedures. 2.4. RT-PCR To confirm the gene disruption by RT-PCR, total RNA from wild type or DJ-1−/− cells was extracted using Sepasol-RNA I Super (Nacalai Tesque). First-strand cDNA synthesis was performed using SuperScript III First-Strand Synthesis System for RT-PCR (Life Technologies). All procedures were performed according to manufacturer’s protocols. The subsequent PCR was performed using primers 5 -ATCAAGGTGACTGTTGCAGG-3 and 5 GTGCCTTCACTTGTTCAGCC-3 , which hybridize sequences in exon 3 and 7 respectively. 2.5. Western blotting
2. Materials and methods 2.1. Cell culture DT40 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% chicken serum, 100 units/ml penicillin and 100 g/ml streptomycin at 39 ◦ C with 5% CO2 . 2.2. Generation of DJ-1−/− DT40 Cells DJ-1 gene disruption constructs (DJ-1-puro, DJ-1-his) were generated from genomic PCR products combined with puromycinor histidinol-resistant selection cassettes flanked by loxP sites using MultiSite Gateway technology (Invitrogen). All procedures were performed according to the manufacturer’s instructions. The drug resistant cassettes were inserted in exon 4 of DJ-1 gene. Genomic DNA sequences of wild type DT40 cells were amplified using primers 5 -GGGGACAACTTTGTATAGAAAAGTTGATGGCCTCGAAAAGAGCGTTGGTGATTCTG-3 and 5 -GGGGACTGCTTTTTTGTACAAACTTGCCAGGCAGGACAATGACATC-3 for the 5 -arm and 5 -GGGGACAGCTTTCTTGTACAAAGTGGAGGTAACCTTGGAGCTCAGA-3 and 5 -GGGGACAACTTTGTATAATAAAGTTGGCAATCCCTGTGTAAGGCAG-3 for the 3 -arm. To generate the 5 - and 3 -arm entry clones, 1.5 kb from the 5 - and 4.4 kb from the 3 -arm were subcloned by BP recombination into the donor vectors pDONR P4-P1R and pDONR P2R-P3, respectively. To generate the targeting vector by LR recombination, we used the 5 -arm entry clone, 3 -arm entry clone, pDEST DTA-MLS, and Puro or His entry clones (Iiizumi et al., 2006). To generate DJ-1+/− cells, DJ-1-puro construct was linearized by AscI and transfected into wild type DT40 cells. To generate DJ-1−/− cells, DJ-1-his construct was linearized using AscI and transfected to DJ-1+/− cells. DNA transfection and selection were performed as described previously (Kohzaki et al., 2010). Briefly, 10–30 g of linearized targeting vectors was electroporated into 107 DT40 cells with Gene Pulser apparatus (Bio-Rad
To confirm the absence of DJ-1 protein in DJ-1−/− cells by Western blotting, cells were lysed on ice with 2× lysis buffer (0.125 M Tris–HCl, 4% SDS, 10% -mercaptoethanol, 0.02% BPB, 20% glycerol) and boiled for 15 min. SDS-PAGE and immunoblotting were carried out using standard procedures. Rabbit polyclonal anti-DJ-1 antibody was obtained from Novus. Rabbit polyclonal anti-GAPDH antibody was obtained from Sigma. 2.6. Measurement of intracellular reactive oxygen species (ROS) accumulation Wild type or DJ-1−/− DT40 cells were treated with 10 M hydrogen peroxide (H2 O2 ) for 24 h and monitored for intracellular ROS accumulation by labeling with 5-(and-6)-carboxy2 ,7 -dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA, Life Technologies) according to the manufacturer’s protocol with slight modification. Briefly, cells were incubated in 5 M carboxyH2 DCFDA in PBS with 1 mM CaCl2 and 0.5 mM MgCl2 for 30 min, followed by being washed once with ice-cold PBS and resuspended in 2 ml of PBS. The fluorescence intensity was measured using a flow cytometer (FACSCalibur, Becton Dickinson). 2.7. Analysis of hydrogen-peroxide-induced cell death Wild type or DJ-1−/− DT40 cells were treated with 10 M H2 O2 for 24 h and analyzed for apoptotic marker and plasma membrane integrity with Annexin-V and propidium iodide staining according to the manufacturer’s protocol (Roche Applied Science). Briefly, cells were washed once with PBS and incubated with labeling solution containing both Annexin-V and propidium iodide for 15 min at 25 ◦ C. Fluorescence intensity was measured using a flow cytometer (FACSCalibur, Becton Dickinson). The results were analyzed using CellQuest software. Gating was determined empirically in one experiment and applied to all subsequent experiments.
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2.8. Measurement of mitochondria membrane potential Mitochondria membrane potential was analyzed using MitoTracker Green FM, MitoTracker Red CMXRos (Life Technologies) and tetramethylrhodamine ethyl ester (TMRE, Life Technologies). Cells were stained for 30 min with 50 nM MitoTracker Red CMXRos, replated onto a poly-d-lysine coated chambered coverglass (Nunc) and imaged using an inverted confocal microscope (LSM510 Meta, Zeiss). As a positive control for mitochondria membrane potential loss, DT40 cells were incubated with 10 M of carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 60 min, stained with MitoTracker Red CMXRos and imaged as described above. Flow cytometric analysis was performed using cells stained with 100 nM TMRE or 25 nM MitoTracker Green FM for 30 min. Analysis was performed using FACSCalibur (Becton Dickinson) and CellQuest software. At least 10,000 cells were analyzed for the corresponding fluorescence in each experiment. Gating was determined empirically in one experiment and applied to all subsequent experiments. 2.9. Analysis of mitochondrial morphology by immunostaining DT40 cells were attached onto a poly-d-lysine coated coverslip for 1 h at 39 ◦ C with 5% CO2 . The coverslips were gently immersed into 4% paraformaldehyde in PBS for 15 min for fixation and then into 0.1% NP-40 in PBS for 5 min for permeabilization. After blocking with 1% BSA in PBS for 30 min, the samples were incubated with polyclonal anti-Tom20 antibody (SantaCruz) in 1% BSA/PBS at 4 ◦ C overnight. The samples were then stained with Alexa Fluor 568 (Life Technologies) in 1% BSA/PBS for one hour at 25 ◦ C and mounted with Vectashield mounting media with DAPI (Vector Laboratories). Images were randomly obtained using an inverted confocal microscope (LSM510 Meta; Zeiss) and analyzed by Spot Detector Version 3.0 BioApplication (Thermo Fisher Scientific). The size of each spot surrounded by Tom20-positive signal was analyzed. The number of the spots per cell was also counted. The average size of spot was determined for 113 cells in wild type and 114 cells in DJ-1−/− cells.
has been suggested to have multiple possible functions, a consistent finding is that DJ-1 protects against oxidative stress in vitro and in vivo (Cookson, 2010). Therefore, we first investigated whether this was recapitulated in DT40 cells. We treated cells with H2 O2 and determined intracellular accumulation of ROS by flow cytometric analysis with 5-(and-6)-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA). DJ-1−/− DT40 cells showed significantly increased accumulation of intracellular ROS compared to wild type cells (Fig. 2A). To examine vulnerability of DJ-1−/− DT40 cells to oxidative stress, we analyzed H2 O2 -induced cell death. At 24 h after H2 O2 treatment, we measured the ratio of viable cells, early-apoptotic cells, and late-apoptotic and necrotic cells by flow cytometric analysis. We found that the ratio of viable cells was significantly lower and that early-apoptotic and late-apoptotic and necrotic cells were significantly higher in DJ-1−/− cells than in wild type cells (Fig. 2B and C). These results were compatible with the notion that DJ-1 has physiological antioxidant property in DT40 cells. 3.3. Decreased mitochondrial membrane potential in DJ-1−/− DT40 cells Mitochondrial dysfunction has long been implicated in the etiopathogenesis of PD (Imai and Lu, 2011). Indeed, DJ-1-deficient mammalian cells show mitochondrial dysfunction (Krebiehl et al., 2010). To analyze whether loss of DJ-1 resulted in mitochondrial dysfunction in DT40 cells, we evaluated mitochondrial membrane potential (MMP) using the following two MMP-dependent dyes, MitoTracker Red CMXRos and tetramethylrhodamine ethyl ester
2.10. Statistical analysis In phenotype analyses, experiments were repeated independently three times using one representative DJ-1−/− clone after having confirmed by a preliminary experiment that two or three different DJ-1−/− clones exhibit the same phenotype. GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA) was used for all statistical analyses. Statistical tests are described in the figure legends. 3. Results 3.1. Generation of DJ-1−/− DT40 Cells We disrupted two DJ-1 alleles by sequentially transfecting the two targeting constructs (DJ-1-puro, DJ-1-his) carrying puromycin or histidinol resistance gene into DT40 cells (Fig. 1A). Disruption of both alleles was confirmed by genomic Southern blotting (Fig. 1B). Absence of detectable mRNA or protein was confirmed by RT-PCR using primers designed to flank drug-resistance gene (Fig. 1C) and western blotting (Fig. 1D). 3.2. Increased vulnerability to oxidative stress in DJ-1−/− DT40 cells DJ-1 was originally identified as an oncogene and was found to be the causative gene of autosomal recessive Parkinson’s disease, PARK7 (Nagakubo et al., 1997; Bonifati et al., 2003). Although DJ-1
Fig. 1. Targeted disruption of DJ-1 in DT40 cells. (A) Schematic representation of the chicken DJ-1 locus and the gene-targeting constructs. Black squares indicate exon 2–7. The restriction enzyme sites and the probe used for genomic Southern blot analysis are shown. (B) Southern blot analysis of BglII-digested genomic DNA from wild type, DJ-1+/−, and DJ-1−/− DT40 cells. (C) Representative RT-PCR analysis of wild type and DJ-1−/− cells. The forward primer was designed in exon 3, and the reverse primer was designed in exon 7. The expected length of amplicon from wild-type DT40 cells is 460 bp. No band corresponding to DJ-1 was observed in DJ-1 knockout cells. (D) Western blot analysis of whole cell lysate from wild type and DJ-1−/− DT40 cells. Blots were probed with anti-DJ-1 polyclonal antibody and anti-GAPDH polyclonal antibody. No band corresponding to DJ-1 was observed.
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Fig. 2. Increased vulnerability to oxidative stress in DJ-1 −/− DT40 cells. (A) Flow cytometric analysis of intracellular ROS. Wild type or DJ-1 −/− DT40 cells were treated with 10 M H2 O2 for 24 h and stained with carboxy-H2 DCFDA, a ROS indicator. The fluorescence intensity was measured by flow cytometry. The amount of intracellular ROS after H2 O2 treatment was significantly greater in DJ-1 −/− cells. Error bars indicate mean ± SEM values from three independent experiments. *P < 0.05, N.S.; not significant by Bonferroni multiple comparison test. (B) Representative flow cytometric analysis of cell viability after H2 O2 treatment. Wild type or DJ-1 −/− DT40 cells were treated with 10 M H2 O2 for 24 hours and stained with Annexin-V (AV) and propium iodide (PI). Lower right quadrant (AV positive and PI negative) corresponds to early-apoptotic cells, and upper right quadrant (AV and PI doublepositive) corresponds to late-apoptotic and necrotic cells. Cells not stained with AV and PI (lower left quadrant) are viable cells. (C) Quantification of viable, early-apoptotic, and late-apoptotic and necrotic cells after treatment with 10 M H2 O2 for 24 h. The percentage of the cells in lower left (LL), lower right (LR) or upper right (UR) quadrant shown in (B) were quantified and were analyzed by Bonferroni multiple comparison test. Error bars indicate mean ± SEM values from three independent experiments. *P < 0.05, N.S.; not significant.
(TMRE). The staining with the former reagent showed a decrease of MMP in DJ-1−/− DT40 cells, as indicated by lower levels of staining in the mutant cells compared to wild type DT40 cells (Fig. 3A). We confirmed this result by detecting the intensity of TMRE staining, a quantitative indicator of membrane potential, by flow cytometry. The MMP of DJ-1−/− DT40 cells was significantly decreased compared to wild type cells (Fig. 3B and C). To exclude the possibility that the decreased staining with these dyes was caused by a decrease in the number of mitochondria, we stained the cells with MitoTracker Green, which is a MMP-independent dye. The intensity of MitoTracker Green signals did not differ between wild type and DJ-1−/− cells. This indicated that the reduced staining with MitoTracker Red CMXRos or TMRE was not due to decrease in the number of mitochondria in DJ-1−/− cells (Fig. 3D). Overall, these results suggested that loss of DJ-1 in DT40 cells resulted in decreased mitochondrial membrane potential. 3.4. Fragmented mitochondria in DJ-1−/− DT40 cells Recent studies have consistently demonstrated that DJ-1deficient mammalian cells exhibit mitochondrial fragmentation (Krebiehl et al., 2010; Irrcher et al., 2010). To confirm the reproducibility of this phenotype in DJ-1−/− DT40 cells, we immunostained mitochondria with Tom20, a mitochondrial outer membrane specific protein, and imaged by laser confocal microscope (Fig. 4A). We determined the average size of mitochondria using Spot Detector software by analyzing the size of each spot surrounded by Tom20-positive signal and the number of the spots per cell. Remarkably, in DJ-1−/− DT40 cells, the average size of these spots was significantly decreased (Fig. 4B) and the number of these spots per cell was significantly increased (Fig. 4C) compared to wild type DT40 cells. 4. Discussion In the present study, we generated DJ-1 deficient DT40 cells, which exhibited vulnerability to oxidative stress, mitochondrial dysfunction and mitochondrial fragmentation. We utilized DT40, a lymphocyte cell line, as a novel PD cellular model. The rationale for our approach is that Parkin, PINK1 and DJ-1 are highly conserved across species and are expressed in lymphocytes. Moreover, analysis of lymphocytes would contribute to
understanding the pathomechanism of PD. This is evidenced by subclinical PD-related phenotypes in lymphocytes or lymphoblasts of PD patients. Mitochondrial dysfunction and increased oxidative stress were observed in lymphocytes of PD patients (Müftüoglu et al., 2004; Prigione et al., 2009). Lymphoblasts established from a PD patient with DJ-1 mutation exhibited abnormal mitochondrial fragmentation (Irrcher et al., 2010). These subclinical phenotypes are consistent with the phenotype of DJ-1-deficient DT40 cells. These lines of evidence support the recent concept of PD as a systemic disorder (Imai and Lu, 2011) and demonstrates the validity of our approach. Although the effect of DJ-1 loss in DT40 cells on ROS accumulation and mitochondrial membrane potential were relatively small compared to previous studies, we speculate that this was because of the highly rapid turnover of DT40 cells. Furthermore, although the mitochondrial morphology is relatively fragmented in wild type DT40 cells compared to those typically observed in adherent culture cell lines, the difference in the quantitated Tom20-positive area indicated that loss of DJ-1 led to further mitochondrial fragmentation in DT40 cells. The advantage of this approach compared to the currentlyavailable cellular models including induced pluripotent stem cells-derived lines from patients, mouse embryonic fibroblasts from knockout mice and RNAi approaches, are the efficacy and consistency in generating and analyzing the effect of multiple gene disruption. These advantages rely on the following multiple unique characteristics of this cell line. Firstly, DT40 cell line exhibits the extraordinary high ratio of targeted to random DNA integration (Buerstedde and Takeda, 1991). This ensures highly efficient multiple targeted gene disruption in a single cell using seven different selection markers, which can be recycled by the cre/loxP (Winding and Berchtold, 2001; Arakawa et al., 2001). Secondly, although RNAi technology has been widely used in mammalian cell lines for targeted knockdown, the residual mRNA is always problematic for interpretation of the resulting phenotype (Neumann et al., 2010). Genetically engineered knockout of DT40 helps overcome this problem. The fact that wild type and gene-disrupted DT40 cells have completely identical genetic background also contributes to increase consistency in phenotype analysis. Moreover, DT40 cells have a short doubling time (∼8 h) and are genetically stable even after repeated passages compared to mammalian cells including mouse embryonic fibroblasts. These also help increase efficiency and consistency in phenotype analysis.
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Fig. 4. Fragmented mitochondria in DJ-1−/− DT40 cells. (A) Representative imaging of wild type and DJ-1−/− DT40 cells immunostained with anti-Tom20 antibody. Mitochondria were fragmented in DJ-1−/− cells. (B) Quantification of mitochondrial size. Randomly obtained images from anti-Tom20-stained DT40 cells were analyzed by Spot Detector Version 3.0 BioApplication. The average size of Tom20-positive spots was significantly smaller in DJ-1−/− cells. Error bars indicate mean ± SEM values. *P < 0.05 by unpaired t-test. (C) Quantification of mitochondrial number. The number of Tom20-positive spots per cell was significantly larger in DJ-1−/− cells. Error bars indicate mean ± SEM values. *P < 0.05 by unpaired t-test.
Fig. 3. Decreased mitochondrial membrane potential in DJ-1−/− DT40 cells. (A) Representative imaging of wild type and DJ-1−/− DT40 cells stained with MitoTracker Red CMXRos. DJ-1−/− cells had lower staining intensity of mitochondria than wild-type cells. CCCP treatment was performed as a positive control. (B) Representative flow cytometric analysis of TMRE staining intensity. (C) Quantification of TMRE staining. DJ-1−/− cells had significantly lower TMRE staining. Three independent experiments were performed and analyzed by unpaired t-test. Error bars indicate mean ± SEM values. *P < 0.01. (D) Quantification of MitoTracker GreenFM staining from flow cytometric analysis. Three independent experiments were performed and analyzed by unpaired t-test. Error bars indicate mean ± SEM values. N.S., not significant.
All these features make DT40 cell line attractive for analyzing effects of gene disruption as well as functional relationships among multiple genes. These advantages of DT40 cell lines have been previously taken to analyze the functions of B cell antigen receptor signaling, histone gene function, RNA processing, DNA repair, cell cycle, calcium signaling and autophagy (Winding and Berchtold, 2001; Alers et al., 2011). As accumulating evidence suggests the primary role of mitochondrial dysfunction in the pathomechanism of PD (Schapira, 2012), it is crucial to evaluate mitochondria function in PD models. We showed that mitochondrial membrane potential and morphology are available as a readout of phenotypic analysis in DT40 cells. Generation and functional analyses of double- or tripleknockout DT40 cell model of Parkin, PINK1 and DJ-1 will help further reveal the functional association among these genes in the maintenance of mitochondria as well as in the pathophysiology of PD. Furthermore, elucidating the pathomechanism of PD using DT40 lymphocyte line could lead to the development of novel tools to utilize patients’ lymphocytes as noninvasive diagnostic or therapeutic biomarkers. This approach also has a great potential for developing high-throughput assays for drug screening using patient-derived lymphocytes. Conflict of interest The authors have no conflict of interest to declare.
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