- Email: [email protected]
Batten disease is linked to altered expression of mitochondria-related metabolic molecules
Neurochemistry International 62 (2013) 931–935
Contents lists available at SciVerse ScienceDirect
Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Batten disease is linked to altered expression of mitochondria-related metabolic molecules Sunyang Kang a,1, Jae Hong Seo b,1, Tae-Hwe Heo b,⇑, Sung-Jo Kim a,⇑ a b
Department of Biotechnology, Hoseo University, 165 Baebang, Asan, Chungnam, Republic of Korea Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
a r t i c l e
i n f o
Article history: Received 17 January 2013 Received in revised form 1 March 2013 Accepted 12 March 2013 Available online 21 March 2013 Keywords: Batten disease Mitochondria ROS
a b s t r a c t Batten disease (BD)—also known as juvenile neuronal ceroid lipofuscinoses—is an inherited neurodegenerative disorder caused by CLN3 gene mutations. Although CLN3-related oxidative and mitochondrial stresses have been studied in BD, the pathologic mechanism of the disease is not clearly understood. To address the molecular factors linked to high levels of oxidative stress in BD, we examined the expression of mitochondria-related metabolic molecules, including pyruvate dehydrogenase (PDH), ATP citrate lyase (ACL), and phosphoenolpyruvate carboxykinase (PEPCK), as well as the apoptosis-related ganglioside, acetyl-GD3. We observed an increased expression of PDH and a decreased expression of ACL, PEPCK, and acetyl-GD3 in BD lymphoblast cells compared to normal cells, possibly resulting in the high ROS levels, mitochondrial membrane depolarization, and apoptosis typically found in BD. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Batten disease, also known as juvenile neuronal ceroid lipofuscinosis (JNCL), is an inherited disorder characterized by a progressive neurodegeneration that usually appears by the ages of 5–7 and is caused by mutations in the CLN3 gene (The International Batten Disease Consortium, 1995). The function of the corresponding CLN3 protein is not fully understood; however, several studies have suggested that it is closely linked to oxidative stress (Kim et al., 2010; Tuxworth et al., 2011; Yoon et al., 2011). Using a Drosophila model, it has been demonstrated that CLN3 plays a role as a defensive component against oxidative stress-induced neuronal cell death (Tuxworth et al., 2011). Oxidative stress may be the result of metabolic defects in CLN3 leading to increased reactive oxygen species (ROS) production (Benedict et al., 2007). Mitochondria could be a source, sink, or target of ROS and perform numerous other tasks regulating cell viability (Starkov, 2008). Indeed, ROS production by mitochondria can lead to oxidative damage to metabolic molecules and functions, including the tricarboxylic acid (TCA) cycle, and apoptosis of cells via the release of cytochrome c to the cytosol (Murphy, 2009).
⇑ Corresponding authors. Tel.: +82 2 2164 4053; fax: +82 2 2164 4059 (T.-H. Heo), tel.: +82 41 540 5571; fax: +82 41 548 6231(S.-J. Kim). E-mail addresses: [email protected] (T.-H. Heo), [email protected] (S.-J. Kim). 1 Co-first authors. 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.03.007
To date, approximately 10 mitochondrial ROS-producing systems have been identified, and among them, TCA cycle enzyme complexes, such as pyruvate dehydrogenase (PDH), have been implicated as mitochondrial ROS sources (Circu and Aw, 2010; Murphy, 2009; Starkov, 2008). PDH catalyzes the oxidation of pyruvate to acetyl-CoA and functionally links glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondrion (Stacpoole, 2012). In the cytosol, acetyl-CoA can be generated from glucose-derived citrate by ATP citrate lyase (ACL) (Watson et al., 1969) and is required for acetylation reactions that modify proteins such as histones (Chypre et al., 2012). ACL also catalyzes the formation of oxaloacetate from cytosolic citrate while simultaneously hydrolyzing ATP (Chypre et al., 2012). ACL-produced oxaloacetate can be reduced to malate, which returns to the mitochondria, or converted to phosphoenolpyruvate, which is catalyzed by another mitochondrial-related cytosolic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). Altered expression of PEPCK, a gluconeogenic enzyme, has been purported to be associated with oxidative stress (Ito et al., 2006). Increased ceramide levels in the brain have been reported in different BD types (Puranam et al., 2007). Ceramide is generated in early apoptosis via hydrolysis of the membrane phospholipid, sphingomyelin (Kolesnick and Krönke, 1998), and then converted into gangliosides (Rippo et al., 2000). GD3 is a ceramide-derived glycosphingolipid (ganglioside), and it induces apoptosis by targeting mitochondria (Jana et al., 2009; Rippo et al., 2000). GD-induced mitochondrial alterations result in the loss of membrane potential, the activation of caspase-9, and ROS production (De Maria, 1997; Rippo et al., 2000). Proapoptotic GD3 becomes ineffective when
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Fig. 1. Elevated levels of ROS and death rates in BD cells. (A) BD and normal cells were treated with DCF-DA and were subjected to flow cytometry analysis to assess ROS levels, as measured with DCF-DA fluorescence. Mean and standard deviation MFI values of triplicate measurements were determined, and statistical significance was evaluated using Student’s t-test (p < 0.05). (B) BD and normal cells were treated with ViaCount reagent and were run on a Guava easyCyte flow cytometer. Mean and standard deviation values of triplicate measurements were determined, and statistical significance was evaluated using Student’s t-test (p < 0.05).
O-acetylation of GD3 sialic acid (9-O-acetyl-GD3) occurs (Malisan et al., 2002). O-Acetylation changes the structural properties, antigenic specificities, and biological activities of sialic acid (Varki, 1997). In this report, we examined the expression of mitochondria-related metabolic molecules to identify possible contributors to the high levels of ROS and death rate in BD cells.
2. Materials and methods 2.1. Cell lines and culture conditions The cells used in this study were lymphoblast cell lines from normal controls (American Type Culture Collection, Rockville, MD, USA) and from patients with BD (Coriell Institute for Medical
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Research). Cells were cultured in RPMI-1640 medium (Cat#: A10491-01, Invitrogen, Carlsbad, CA, USA) and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gemcell; Gemini Bio Products, West Sacramento, CA, USA), 100 unit/ml penicillin-streptomycin (Hyclone, Logan, UT, USA), and 1% L-glutamine (Well Gene Inc., Seoul, South Korea) in 100-mm dishes (SPL Life Science, Seoul, South Korea) in a CO2 incubator (Thermo Scientific) at 1 106 cells/ml. 2.2. ROS assay ROS levels were detected using 20 ,70 -dichlorofluorescein diacetate (DCF-DA) (Sigma). In brief, 1 105 BD and normal cells were washed with PBS (5% FBS) and incubated with 10 lM DCF-DA in RPMI-1640 with 10% FBS at 37 °C for 15 min in 5% CO2. Afterwards, the cells were trypsinized, run on a Guava easyCyte flow cytometer, and analyzed by Guava InCyte software (Millipore, Billerica, MA, USA). 2.3. Cell viability assay The ViaCount kit was purchased from Guava Technologies and used in coordination with the Guava Personal Cytometry system. The ViaCount kit allows for the identification of dead cells. BD cells (1 106) and normal cells were added to 380 ll of ViaCount reagent with the addition of the cationic dye JC-1 (5 lg/ml) and incubated for 5 min. Samples were run on a Guava easyCyte flow cytometer and analyzed using ViaCount software (Millipore).
2.4. Western blot analysis BD (1 106) and normal cells were cultured on 100-mm plates in medium at 37 °C in an incubator containing 95% air and 5% CO2. After 24 h, cells were collected and analyzed. Proteins were extracted using RIPA buffer with a protease-inhibitor cocktail (Sigma). From each sample, 30 lg of protein was removed, electrophoresed on a sodium dodecyl sulfate–polyacrylamide gel (Bio-Rad), and then transferred to a polyvinylidene fluoride membrane (Bio-Rad) for Western blot analysis. The protein-containing membrane was blocked using 5% skim milk (Bio-Rad) and then incubated with primary anti-ACL, anti-PEPCK, anti-PDH (Santa Cruz Biotechnology, CA, USA), and anti-b-actin (Sigma) antibodies. Subsequently, anti-goat, anti-rabbit, and anti-mouse (Santa Cruz Biotechnology) secondary antibodies were incubated with the membrane, and the protein band was then visualized using Super-Signal West Pico Luminal/Enhancer solution (Pierce). Quantification of band intensities was carried out by TINA 2.0 densitometric analytical system (Raytest, Isotopenmesgerate GmbH, Germany). Experiments were performed in triplicate. Statistical significance was evaluated by Student’s t-test. P values less than 0.05 were considered statistically significant. 2.5. Thin layer chromatography (TLC) analysis The lipids were extracted using a chloroform:methanol (1:2 v/ v) extraction method reported by Bligh and Dyer. Gangliosides of sphingolipids were separated using a TLC silica gel 60 plate (Merck,
Normal
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PDH Ⴋ β-actin Ⴋ
PDH / Actin ratio
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5 4 3 2 1 0
∗
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Normal
BD
BD
Acetyl -GD3 ganglioside Non -acetyl GD3 ganglioside Fig. 2. Altered expressions of mitochondria-related metabolic molecules in BD cells. (A) Western blot analysis for PDH, ACL, and PEPCK proteins in cultured BD and normal cells. b-actin was used as a loading control. Densitometric analysis of each protein was performed by a protein to b-actin ratio (each result represents the mean ± SD of three experiments performed in triplicate). (B) Acetyl-GD3 ganglioside and GD3 ganglioside in BD or normal cells were analyzed by TLC.
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Germany), and separations were performed in a horizontal TLC chamber with an 18-cm separation length using a solution of chloroform:methanol:0.22% aqueous CaCl2 (60:35:8 v/v/v). TLC plates were visualized by spraying with H2SO4, followed by heating at 130 °C for 30 min.
2.6. Mitochondrial depolarization analysis This assay was performed using the MitoPotential Kit according to the manufacturer protocol (Guava easyCyte). In brief, the cells were harvested by trypsinization, washed with PBS (5% FBS), loaded with the cationic dye JC-1 for 30 min, and analyzed using the Guava easyCyte flow cytometer with MitoPotential software (Millipore). In non-apoptotic cells, the JC-1 dye enters the mitochondrial matrix as aggregates and stains the mitochondria orange. In apoptotic cells, the mitochondrial membrane potential collapses, and the JC-1 enters the cytoplasm as a monomeric form where it fluoresces green.
3. Results 3.1. BD cells show higher ROS levels and death rates than normal cells Because the function of CLN3 protein has been proposed to be associated with oxidative stress, we examined ROS levels in BD cells and compared them with normal control cells by using a detector of ROS, DCF-DA. ROS accumulated more in BD cells than in normal cells (Fig. 1A). Because high levels of ROS may influence the viability of cells, we determined and compared apoptotic percentages of normal and BD cells. BD cells showed an approximately 3-fold higher death rate than normal cells (Fig. 1B).
3.2. BD cells show an expression profile of mitochondria-related metabolic molecules different from normal cells Observations of elevated ROS prompted us to examine the expression levels of metabolic enzymes linked to ROS production. Initially, we detected pyruvate dehydrogenase (PDH), the entry enzyme of TCA cycle, by Western blot and found that its expression level was significantly higher in BD cells compared to normal cells (Fig. 2A). An elevated TCA cycle rate caused by the overexpression of PDH may lead to the production of large amounts of citrate and the migration of mitochondrial citrate into the cytosol. Cytosolic citrate metabolism was measured by detecting ATP citrate lyase (ACL), a key enzyme that catalyzes the conversion of citrate into cytosolic acetyl-CoA and oxaloacetate. In contrast to PDH, the expression level of ACL was lower in BD cells than in normal cells (Fig. 2A). Reduced ACL may lead to decreased production of cytosolic acetyl-CoA, which is the requisite building block for acetylation reactions that modify proteins and lipids. Indeed, the level of acetyl-GD3 ganglioside, the inactive form of proapoptotic GD3 ganglioside, was lower, and apoptotic GD3 was higher in BD cells than in normal cells (Fig. 2B). ACL-generated oxaloacetate can be reduced to malate, which returns to the mitochondria or is converted to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PEPCK). PEPCK expression levels were measured and found to be slightly decreased (Fig. 2A).
3.3. The percentage of mitochondrial membrane-depolarized cells is higher in BD cells than in normal cells Next, mitochondrial potential was evaluated and compared in BD and normal cells to verify whether high ROS levels induce mitochondrial dysfunction. The depolarization of the mitochondrial
Fig. 3. Mitochondrial membrane depolarization in BD cells. Membrane potential of mitochondria was analyzed by MitoPotential Kit according to the manufacturer protocol (Guava easyCyte). In brief, the cells were loaded with the cationic dye JC-1 and analyzed by Guava easyCyte flow cytometer using MitoPotential software (Millipore).
S. Kang et al. / Neurochemistry International 62 (2013) 931–935
membrane was evaluated through the measurement of JC-1 dye by utilizing a flow cytometry-based assay. The percentage of membrane-depolarized cells was significantly higher in BD cells than in normal cells (57.9% vs. 23.5%; Fig. 3). 4. Discussion Several sources have implicated oxidative stress in the pathology of Batten disease in yeast (Osório et al., 2007; Vitiello, 2008), Drosophila (Tuxworth et al., 2011), and mouse models (Benedict et al., 2007). Here, we report that high levels of ROS and apoptosis in BD patient cells (Fig. 1) are possibly due to the altered expression of mitochondria-related metabolic molecules resulting in overstimulated TCA cycles and mitochondrial dysfunction. PDH involved in the TCA cycle has been implicated as a source of ROS in the mitochondria (Murphy, 2009), but little information is available in cases involving BD. We have shown for the first time that PDH is overexpressed by more than three times in BD cells compared to normal cells (Fig. 2). Because PDH is a critical rate-determining enzyme of the TCA cycle responsible for mediating the oxidation of pyruvate to acetyl-CoA and functionally linking glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondrion, overexpression of PDH could lead to overstimulation of the TCA cycle and overproduction of ROS. An overstimulated TCA cycle can be rescued by the consumption of product, such as cytosolic citrate, by ACL, which catalyzes the conversion of citrate into acetyl-CoA and oxaloacetate. However, reduced expression of ACL in BD cells (Fig. 2A) may cause shortages of acetyl-CoA and oxaloacetate and unsuccessfully correct the abnormality of the TCA cycle. The reduced generation of acetyl-CoA by ACL could lead to insufficient acetylation reactions that modify proapoptotic GD3 into anti-apoptotic acetyl-GD3. Indeed, the level of acetylGD3 ganglioside is lower, whereas that of GD3 is higher in BD cells than in normal cells (Fig. 2B). High ROS, high GD3, and low acetylGD3 levels are possible causes of the higher percentage of mitochondrial membrane-depolarized cells in BD (Fig. 3). ACL-produced oxaloacetate can be converted to phosphoenolpyruvate by PEPCK, which prohibits the reduced form of oxaloacetate, malate, from entering into the TCA cycle again. This anti-TCA cyclic activity of PEPCK is downregulated in BD cells. The function of CLN3 remains unclear especially regarding mitochondrial association. Considering that CLN3-mutated Drosophila exhibited a hypersensitive accumulation of ROS against oxidative stress (Tuxworth et al., 2011), the CLN3 protein may directly or indirectly influence the mitochondrial metabolism involving ROS production. Taken together, this report highlights a novel finding that high levels of ROS and apoptosis in BD cells are possibly attributed to the high expression of PDH and low expression of ACL, PEPCK, and acetyl-GD3.
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Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (2010-0002816). This research was supported by Research Fund 2011 of The Catholic University of Korea. References Benedict, J.W., Sommers, C.A., Pearce, D.A., 2007. Progressive oxidative damage in the central nervous system of a murine model for juvenile Batten disease. J. Neurosci. Res. 85, 2882–2891. Chypre, M., Zaidi, N., Smans, K., 2012. ATP-citrate lyase: a mini-review. Biochem. Biophys. Res. Commun. 422, 1–4. Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic. Biol. Med. 48, 749–762. The International Batten Disease Consortium, 1995. Isolation of a novel gene underlying Batten disease, CLN3. Cell 82, 949–957. De Maria, R., 1997. Requirement for GD3 ganglioside in CD95- and ceramideinduced apoptosis. Science 277, 1652–1655. Ito, Y., Oumi, S., Nagasawa, T., Nishizawa, N., 2006. Oxidative stress induces phosphoenolpyruvate carboxykinase expression in H4IIE cells. Biosci. Biotechnol. Biochem. 70, 2191–2198. Jana, A., Hogan, E.L., Pahan, K., 2009. Ceramide and neurodegeneration: susceptibility of neurons and oligodendrocytes to cell damage and death. J. Neurol. Sci. 278, 5–15. Kim, S.-J., Zhang, Z., Saha, A., Sarkar, C., Zhao, Z., Xu, Y., Mukherjee, A.B., 2010. Omega-3 and omega-6 fatty acids suppress ER- and oxidative stress in cultured neurons and neuronal progenitor cells from mice lacking PPT1. Neurosci. Lett. 479, 292–296. Kolesnick, R.N., Krönke, M., 1998. Regulation of ceramide production and apoptosis. Annu. Rev. Physiol. 60, 643–665. Malisan, F., Franchi, L., Tomassini, B., Ventura, N., Condo, I., Rippo, M.R., Rufini, A., Liberati, L., Nachtigall, C., Kniep, B., Testi, R., 2002. Acetylation suppresses the proapoptotic activity of GD3 ganglioside. J. Exp. Med. 196, 1535–1541. Murphy, M.P., 2009. How mitochondria produce reactive oxygen species. Biochem. J. 417, 1. Osório, N.S., Carvalho, A., Almeida, A.J., Padilla-Lopez, S., Leão, C., Laranjinha, J., Ludovico, P., Pearce, D.A., Rodrigues, F., 2007. Nitric oxide signaling is disrupted in the yeast model for Batten disease. Mol. Biol. Cell 18, 2755–2767. Puranam, K., Qian, W.H., Nikbakht, K., Venable, M., Obeid, L., Hannun, Y., Boustany, R.M., 2007. Upregulation of Bcl-2 and elevation of ceramide in Batten disease. Neuropediatrics 28, 37–41. Rippo, M.R., Malisan, F., Ravagnan, L., Tomassini, B., Condo, I., Costantini, P., Susin, S.A., Rufini, A., Todaro, M., Kroemer, G., Testi, R., 2000. GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion. FASEB J. 14, 2047–2054. Stacpoole, P.W., 2012. The pyruvate dehydrogenase complex as a therapeutic target for age-related diseases. Aging Cell 11, 371–377. Starkov, A., 2008. The role of mitochondria in reactive oxygen species metabolism and signaling. Ann. NY Acad. Sci. 1147, 37–52. Tuxworth, R.I., Chen, H., Vivancos, V., Carvajal, N., Huang, X., Tear, G., 2011. The Batten disease gene CLN3 is required for the response to oxidative stress. Hum. Mol. Gen. 20, 2037–2047. Varki, A., 1997. Sialic acids as ligands in recognition phenomena. FASEB J. 11, 248– 255. Vitiello, S., 2008. The yeast model for batten disease: genetic and physical interactions. Available from:. Watson, J.A., Fang, M., Lowenstein, J.M., 1969. Tricarballylate and hydroxycitrate: substrate and inhibitor of ATP: citrate oxaloacetate lyase. Arch. Biochem. Biophys. 135, 209–217. Yoon, D.-H., Kwon, O.-Y., Mang, J.Y., Jung, M.J., Kim, Do.Yeon., Park, Y.K., Heo, T.-H., Kim, S.-J., 2011. Protective potential of resveratrol against oxidative stress and apoptosis in Batten disease lymphoblast cells. Biochem. Biophys. Res. Commun. 414, 49–52.
Contents lists available at SciVerse ScienceDirect
Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Batten disease is linked to altered expression of mitochondria-related metabolic molecules Sunyang Kang a,1, Jae Hong Seo b,1, Tae-Hwe Heo b,⇑, Sung-Jo Kim a,⇑ a b
Department of Biotechnology, Hoseo University, 165 Baebang, Asan, Chungnam, Republic of Korea Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
a r t i c l e
i n f o
Article history: Received 17 January 2013 Received in revised form 1 March 2013 Accepted 12 March 2013 Available online 21 March 2013 Keywords: Batten disease Mitochondria ROS
a b s t r a c t Batten disease (BD)—also known as juvenile neuronal ceroid lipofuscinoses—is an inherited neurodegenerative disorder caused by CLN3 gene mutations. Although CLN3-related oxidative and mitochondrial stresses have been studied in BD, the pathologic mechanism of the disease is not clearly understood. To address the molecular factors linked to high levels of oxidative stress in BD, we examined the expression of mitochondria-related metabolic molecules, including pyruvate dehydrogenase (PDH), ATP citrate lyase (ACL), and phosphoenolpyruvate carboxykinase (PEPCK), as well as the apoptosis-related ganglioside, acetyl-GD3. We observed an increased expression of PDH and a decreased expression of ACL, PEPCK, and acetyl-GD3 in BD lymphoblast cells compared to normal cells, possibly resulting in the high ROS levels, mitochondrial membrane depolarization, and apoptosis typically found in BD. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Batten disease, also known as juvenile neuronal ceroid lipofuscinosis (JNCL), is an inherited disorder characterized by a progressive neurodegeneration that usually appears by the ages of 5–7 and is caused by mutations in the CLN3 gene (The International Batten Disease Consortium, 1995). The function of the corresponding CLN3 protein is not fully understood; however, several studies have suggested that it is closely linked to oxidative stress (Kim et al., 2010; Tuxworth et al., 2011; Yoon et al., 2011). Using a Drosophila model, it has been demonstrated that CLN3 plays a role as a defensive component against oxidative stress-induced neuronal cell death (Tuxworth et al., 2011). Oxidative stress may be the result of metabolic defects in CLN3 leading to increased reactive oxygen species (ROS) production (Benedict et al., 2007). Mitochondria could be a source, sink, or target of ROS and perform numerous other tasks regulating cell viability (Starkov, 2008). Indeed, ROS production by mitochondria can lead to oxidative damage to metabolic molecules and functions, including the tricarboxylic acid (TCA) cycle, and apoptosis of cells via the release of cytochrome c to the cytosol (Murphy, 2009).
⇑ Corresponding authors. Tel.: +82 2 2164 4053; fax: +82 2 2164 4059 (T.-H. Heo), tel.: +82 41 540 5571; fax: +82 41 548 6231(S.-J. Kim). E-mail addresses: [email protected] (T.-H. Heo), [email protected] (S.-J. Kim). 1 Co-first authors. 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.03.007
To date, approximately 10 mitochondrial ROS-producing systems have been identified, and among them, TCA cycle enzyme complexes, such as pyruvate dehydrogenase (PDH), have been implicated as mitochondrial ROS sources (Circu and Aw, 2010; Murphy, 2009; Starkov, 2008). PDH catalyzes the oxidation of pyruvate to acetyl-CoA and functionally links glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondrion (Stacpoole, 2012). In the cytosol, acetyl-CoA can be generated from glucose-derived citrate by ATP citrate lyase (ACL) (Watson et al., 1969) and is required for acetylation reactions that modify proteins such as histones (Chypre et al., 2012). ACL also catalyzes the formation of oxaloacetate from cytosolic citrate while simultaneously hydrolyzing ATP (Chypre et al., 2012). ACL-produced oxaloacetate can be reduced to malate, which returns to the mitochondria, or converted to phosphoenolpyruvate, which is catalyzed by another mitochondrial-related cytosolic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). Altered expression of PEPCK, a gluconeogenic enzyme, has been purported to be associated with oxidative stress (Ito et al., 2006). Increased ceramide levels in the brain have been reported in different BD types (Puranam et al., 2007). Ceramide is generated in early apoptosis via hydrolysis of the membrane phospholipid, sphingomyelin (Kolesnick and Krönke, 1998), and then converted into gangliosides (Rippo et al., 2000). GD3 is a ceramide-derived glycosphingolipid (ganglioside), and it induces apoptosis by targeting mitochondria (Jana et al., 2009; Rippo et al., 2000). GD-induced mitochondrial alterations result in the loss of membrane potential, the activation of caspase-9, and ROS production (De Maria, 1997; Rippo et al., 2000). Proapoptotic GD3 becomes ineffective when
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N
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Fig. 1. Elevated levels of ROS and death rates in BD cells. (A) BD and normal cells were treated with DCF-DA and were subjected to flow cytometry analysis to assess ROS levels, as measured with DCF-DA fluorescence. Mean and standard deviation MFI values of triplicate measurements were determined, and statistical significance was evaluated using Student’s t-test (p < 0.05). (B) BD and normal cells were treated with ViaCount reagent and were run on a Guava easyCyte flow cytometer. Mean and standard deviation values of triplicate measurements were determined, and statistical significance was evaluated using Student’s t-test (p < 0.05).
O-acetylation of GD3 sialic acid (9-O-acetyl-GD3) occurs (Malisan et al., 2002). O-Acetylation changes the structural properties, antigenic specificities, and biological activities of sialic acid (Varki, 1997). In this report, we examined the expression of mitochondria-related metabolic molecules to identify possible contributors to the high levels of ROS and death rate in BD cells.
2. Materials and methods 2.1. Cell lines and culture conditions The cells used in this study were lymphoblast cell lines from normal controls (American Type Culture Collection, Rockville, MD, USA) and from patients with BD (Coriell Institute for Medical
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Research). Cells were cultured in RPMI-1640 medium (Cat#: A10491-01, Invitrogen, Carlsbad, CA, USA) and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gemcell; Gemini Bio Products, West Sacramento, CA, USA), 100 unit/ml penicillin-streptomycin (Hyclone, Logan, UT, USA), and 1% L-glutamine (Well Gene Inc., Seoul, South Korea) in 100-mm dishes (SPL Life Science, Seoul, South Korea) in a CO2 incubator (Thermo Scientific) at 1 106 cells/ml. 2.2. ROS assay ROS levels were detected using 20 ,70 -dichlorofluorescein diacetate (DCF-DA) (Sigma). In brief, 1 105 BD and normal cells were washed with PBS (5% FBS) and incubated with 10 lM DCF-DA in RPMI-1640 with 10% FBS at 37 °C for 15 min in 5% CO2. Afterwards, the cells were trypsinized, run on a Guava easyCyte flow cytometer, and analyzed by Guava InCyte software (Millipore, Billerica, MA, USA). 2.3. Cell viability assay The ViaCount kit was purchased from Guava Technologies and used in coordination with the Guava Personal Cytometry system. The ViaCount kit allows for the identification of dead cells. BD cells (1 106) and normal cells were added to 380 ll of ViaCount reagent with the addition of the cationic dye JC-1 (5 lg/ml) and incubated for 5 min. Samples were run on a Guava easyCyte flow cytometer and analyzed using ViaCount software (Millipore).
2.4. Western blot analysis BD (1 106) and normal cells were cultured on 100-mm plates in medium at 37 °C in an incubator containing 95% air and 5% CO2. After 24 h, cells were collected and analyzed. Proteins were extracted using RIPA buffer with a protease-inhibitor cocktail (Sigma). From each sample, 30 lg of protein was removed, electrophoresed on a sodium dodecyl sulfate–polyacrylamide gel (Bio-Rad), and then transferred to a polyvinylidene fluoride membrane (Bio-Rad) for Western blot analysis. The protein-containing membrane was blocked using 5% skim milk (Bio-Rad) and then incubated with primary anti-ACL, anti-PEPCK, anti-PDH (Santa Cruz Biotechnology, CA, USA), and anti-b-actin (Sigma) antibodies. Subsequently, anti-goat, anti-rabbit, and anti-mouse (Santa Cruz Biotechnology) secondary antibodies were incubated with the membrane, and the protein band was then visualized using Super-Signal West Pico Luminal/Enhancer solution (Pierce). Quantification of band intensities was carried out by TINA 2.0 densitometric analytical system (Raytest, Isotopenmesgerate GmbH, Germany). Experiments were performed in triplicate. Statistical significance was evaluated by Student’s t-test. P values less than 0.05 were considered statistically significant. 2.5. Thin layer chromatography (TLC) analysis The lipids were extracted using a chloroform:methanol (1:2 v/ v) extraction method reported by Bligh and Dyer. Gangliosides of sphingolipids were separated using a TLC silica gel 60 plate (Merck,
Normal
BD
PDH Ⴋ β-actin Ⴋ
PDH / Actin ratio
A
5 4 3 2 1 0
∗
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BD
ACL Ⴋ
ACL / Actin ratio
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1
∗
0.5
0
β-actin Ⴋ
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PEPCK / Actin ratio
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∗
0.5 0
Normal
BD
Normal
BD
BD
Acetyl -GD3 ganglioside Non -acetyl GD3 ganglioside Fig. 2. Altered expressions of mitochondria-related metabolic molecules in BD cells. (A) Western blot analysis for PDH, ACL, and PEPCK proteins in cultured BD and normal cells. b-actin was used as a loading control. Densitometric analysis of each protein was performed by a protein to b-actin ratio (each result represents the mean ± SD of three experiments performed in triplicate). (B) Acetyl-GD3 ganglioside and GD3 ganglioside in BD or normal cells were analyzed by TLC.
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Germany), and separations were performed in a horizontal TLC chamber with an 18-cm separation length using a solution of chloroform:methanol:0.22% aqueous CaCl2 (60:35:8 v/v/v). TLC plates were visualized by spraying with H2SO4, followed by heating at 130 °C for 30 min.
2.6. Mitochondrial depolarization analysis This assay was performed using the MitoPotential Kit according to the manufacturer protocol (Guava easyCyte). In brief, the cells were harvested by trypsinization, washed with PBS (5% FBS), loaded with the cationic dye JC-1 for 30 min, and analyzed using the Guava easyCyte flow cytometer with MitoPotential software (Millipore). In non-apoptotic cells, the JC-1 dye enters the mitochondrial matrix as aggregates and stains the mitochondria orange. In apoptotic cells, the mitochondrial membrane potential collapses, and the JC-1 enters the cytoplasm as a monomeric form where it fluoresces green.
3. Results 3.1. BD cells show higher ROS levels and death rates than normal cells Because the function of CLN3 protein has been proposed to be associated with oxidative stress, we examined ROS levels in BD cells and compared them with normal control cells by using a detector of ROS, DCF-DA. ROS accumulated more in BD cells than in normal cells (Fig. 1A). Because high levels of ROS may influence the viability of cells, we determined and compared apoptotic percentages of normal and BD cells. BD cells showed an approximately 3-fold higher death rate than normal cells (Fig. 1B).
3.2. BD cells show an expression profile of mitochondria-related metabolic molecules different from normal cells Observations of elevated ROS prompted us to examine the expression levels of metabolic enzymes linked to ROS production. Initially, we detected pyruvate dehydrogenase (PDH), the entry enzyme of TCA cycle, by Western blot and found that its expression level was significantly higher in BD cells compared to normal cells (Fig. 2A). An elevated TCA cycle rate caused by the overexpression of PDH may lead to the production of large amounts of citrate and the migration of mitochondrial citrate into the cytosol. Cytosolic citrate metabolism was measured by detecting ATP citrate lyase (ACL), a key enzyme that catalyzes the conversion of citrate into cytosolic acetyl-CoA and oxaloacetate. In contrast to PDH, the expression level of ACL was lower in BD cells than in normal cells (Fig. 2A). Reduced ACL may lead to decreased production of cytosolic acetyl-CoA, which is the requisite building block for acetylation reactions that modify proteins and lipids. Indeed, the level of acetyl-GD3 ganglioside, the inactive form of proapoptotic GD3 ganglioside, was lower, and apoptotic GD3 was higher in BD cells than in normal cells (Fig. 2B). ACL-generated oxaloacetate can be reduced to malate, which returns to the mitochondria or is converted to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PEPCK). PEPCK expression levels were measured and found to be slightly decreased (Fig. 2A).
3.3. The percentage of mitochondrial membrane-depolarized cells is higher in BD cells than in normal cells Next, mitochondrial potential was evaluated and compared in BD and normal cells to verify whether high ROS levels induce mitochondrial dysfunction. The depolarization of the mitochondrial
Fig. 3. Mitochondrial membrane depolarization in BD cells. Membrane potential of mitochondria was analyzed by MitoPotential Kit according to the manufacturer protocol (Guava easyCyte). In brief, the cells were loaded with the cationic dye JC-1 and analyzed by Guava easyCyte flow cytometer using MitoPotential software (Millipore).
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membrane was evaluated through the measurement of JC-1 dye by utilizing a flow cytometry-based assay. The percentage of membrane-depolarized cells was significantly higher in BD cells than in normal cells (57.9% vs. 23.5%; Fig. 3). 4. Discussion Several sources have implicated oxidative stress in the pathology of Batten disease in yeast (Osório et al., 2007; Vitiello, 2008), Drosophila (Tuxworth et al., 2011), and mouse models (Benedict et al., 2007). Here, we report that high levels of ROS and apoptosis in BD patient cells (Fig. 1) are possibly due to the altered expression of mitochondria-related metabolic molecules resulting in overstimulated TCA cycles and mitochondrial dysfunction. PDH involved in the TCA cycle has been implicated as a source of ROS in the mitochondria (Murphy, 2009), but little information is available in cases involving BD. We have shown for the first time that PDH is overexpressed by more than three times in BD cells compared to normal cells (Fig. 2). Because PDH is a critical rate-determining enzyme of the TCA cycle responsible for mediating the oxidation of pyruvate to acetyl-CoA and functionally linking glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondrion, overexpression of PDH could lead to overstimulation of the TCA cycle and overproduction of ROS. An overstimulated TCA cycle can be rescued by the consumption of product, such as cytosolic citrate, by ACL, which catalyzes the conversion of citrate into acetyl-CoA and oxaloacetate. However, reduced expression of ACL in BD cells (Fig. 2A) may cause shortages of acetyl-CoA and oxaloacetate and unsuccessfully correct the abnormality of the TCA cycle. The reduced generation of acetyl-CoA by ACL could lead to insufficient acetylation reactions that modify proapoptotic GD3 into anti-apoptotic acetyl-GD3. Indeed, the level of acetylGD3 ganglioside is lower, whereas that of GD3 is higher in BD cells than in normal cells (Fig. 2B). High ROS, high GD3, and low acetylGD3 levels are possible causes of the higher percentage of mitochondrial membrane-depolarized cells in BD (Fig. 3). ACL-produced oxaloacetate can be converted to phosphoenolpyruvate by PEPCK, which prohibits the reduced form of oxaloacetate, malate, from entering into the TCA cycle again. This anti-TCA cyclic activity of PEPCK is downregulated in BD cells. The function of CLN3 remains unclear especially regarding mitochondrial association. Considering that CLN3-mutated Drosophila exhibited a hypersensitive accumulation of ROS against oxidative stress (Tuxworth et al., 2011), the CLN3 protein may directly or indirectly influence the mitochondrial metabolism involving ROS production. Taken together, this report highlights a novel finding that high levels of ROS and apoptosis in BD cells are possibly attributed to the high expression of PDH and low expression of ACL, PEPCK, and acetyl-GD3.
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