Mol. Cells 2022; 45(7): 454-464
Published online April 21, 2022
https://doi.org/10.14348/molcells.2022.5002
© The Korean Society for Molecular and Cellular Biology
Correspondence to : hjkoh@dau.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
DJ-1 is one of the causative genes of early-onset familial Parkinson’s disease (PD). As a result, DJ-1 influences the pathogenesis of sporadic PD. DJ-1 has various physiological functions that converge to control the levels of intracellular reactive oxygen species (ROS). Based on genetic analyses that sought to investigate novel antioxidant DJ-1 downstream genes, pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to increase survival rates and decrease dopaminergic (DA) neuron loss in DJ-1 mutant flies under oxidative stress. PDK phosphorylates and inhibits the PDH complex (PDC), subsequently downregulating glucose metabolism in the mitochondria, which is a major source of intracellular ROS. A loss-of-function mutation in PDK was not found to have a significant effect on fly development and reproduction, but severely ameliorated oxidative stress resistance. Thus, PDK plays a critical role in the protection against oxidative stress. Loss of PDH phosphatase (PDP), which dephosphorylates and activates PDH, was also shown to protect DJ-1 mutants from oxidative stress, ultimately supporting our findings. Further genetic analyses suggested that DJ-1 controls PDK expression through hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator of the adaptive response to hypoxia and oxidative stress. Furthermore, CPI-613, an inhibitor of PDH, protected DJ-1 null flies from oxidative stress, suggesting that the genetic and pharmacological inhibition of PDH may be a novel treatment strategy for PD associated with DJ-1 dysfunction.
Keywords DJ-1, Drosophila, oxidative stress, Parkinson’s disease, pyruvate dehydrogenase kinase
Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by typical motor symptoms and selective loss of dopaminergic (DA) neurons in the substantia nigra (Lang and Lozano, 1998). Various studies have supported the link between DA neuron loss and oxidative stress originating from excess generation or insufficient elimination of reactive oxygen species (ROS) (Henchcliffe and Beal, 2008). In postmortem brain analyses, oxidative damage to macromolecules, such as DNA, proteins, and lipids, has been shown to be substantially elevated in the substantia nigra of PD patients (Bender et al., 2006; Henchcliffe and Beal, 2008). Various PD animal models have been generated using oxidative stress-inducing agents, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat, and 6-hydroxydopamine (6-OHDA) (Betarbet et al., 2000; Brooks et al., 1999; Langston et al., 1983; Ungerstedt and Arbuthnott, 1970). However, the molecular mechanisms underlying oxidative stress-induced degeneration of DA neurons have not been fully elucidated.
PD mainly develops sporadically, but can also occur due to monogenic mutations (Lesage and Brice, 2009). Among the PD-linked genes, DJ-1 is most closely associated with oxidative stress (Henchcliffe and Beal, 2008). Bonifati et al. (2003) reported that DJ-1 is associated with the development of an autosomal recessive early-onset type of familial PD. Further, subsequent studies revealed that DJ-1 plays various roles in cell biology, such as transcriptional regulation, anti-apoptotic signaling, proteostasis, and mitochondrial regulation in response to oxidative stress (Ariga et al., 2013). DJ-1 is sequentially oxidized at its cysteine residues under oxidative stress, and its activity and subcellular localization are regulated by its oxidative status (Ariga et al., 2013). Notably, excessive oxidation leads to DJ-1 inactivation, and this excessively oxidized form has been identified in the brain of patients with sporadic PD (Bandopadhyay et al., 2004; Choi et al., 2006). Such findings suggest that DJ-1 plays a role in the pathogenesis of both sporadic and familial PD.
In this study, the overexpression of pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to enhance survival rates and ameliorate DA neuron loss in
All flies were grown as described previously (Jang et al., 2020). The
For the oxidative stress assays, three or four groups of 30 3-day-old flies (n = 90 or 120) were starved for 6 h and transferred to a vial containing a gel consisting of phosphate-buffered saline (PBS), 5% sucrose, and an oxidative stress agent (5 mM rotenone or 1% H2O2). To determine the role of
For mitochondrial DNA (mtDNA) polymerase chain reaction (PCR), total DNA was extracted from the thoraces of five 3- and 10-day-old male flies. Quantitative real-time (RT) PCR was performed as described previously (Yang et al., 2017). Rp49 genomic DNA levels were used as an internal control. The results are expressed as fold-changes relative to the control. For the ATP assay, the thoraces of five 3-day-old male flies were dissected, and ATP concentration was measured as previously described (Yang et al., 2017). The relative ATP level was calculated as the ratio of ATP concentration to the total protein concentration. The total protein concentration was determined using the bicinchoninic acid assay (Sigma, USA). The results of the mtDNA PCR and ATP assays are expressed as mean ± SD of three independent experiments.
To investigate the change in the number of DA neurons in
To measure the transactivation activity of Sima on the
Total RNA was extracted from five 3-day-old flies and reverse-transcribed, as previously described (Park et al., 2006). Quantitative RT-PCR was performed using SYBR Premix Ex Taq (Takara, Japan) on QuantStudio 3 (Thermo Fisher Scientific, USA) at the Neuroscience Translational Research Solution Center. Rp49 levels were used as internal controls. The results are expressed as fold-changes relative to the control. The average mRNA levels ± SD were obtained from three independent experiments. The following primer pairs were used: rp49 F (GCT TCA AGA TGA CCA TCC GCC C) and R (GGT GCG CTT GTT CGA TCC GTA AC); PDK F (CTG TTT CCA GTC CGA TTC TCA G) and R (CAG AAG GGC GAT CTC CTT CAT G); prel F (CAC CAG GAA CCT GGG AAT GAC) and R (CAG CAC GTA GTT GAA TCC ATT GGA G); PDP F (CTC CAT ATA GAT GTT CAA GTT CGT G) and R (GTA AAC GAA CTC GTT CTC CCT CAG); and Rab39 F (GAC AGC ACC GTG GGC AAG AG) and R (GAC TTG GTG ATC GAA CGG AAG C).
To quantify DA neurons, four major DA neuron clusters from more than 15 brains of each genotype were observed in a blinded fashion to eliminate bias (n = 30-40). One-way ANOVA with Sidak correction was used to compare three or more groups, while Student’s two-tailed
The central nervous system (CNS) receives most of its energy from oxygen-dependent glucose metabolism. Further, mitochondrial PDC mediates a major step in glucose oxidation, namely the irreversible decarboxylation of pyruvate to acetyl-CoA (Jha et al., 2012). PDC activity is tightly regulated by PDK- and PDP-induced phosphorylation of the PDH E1 subunit, which is the first component of PDC (Park et al., 2018). Recent studies have revealed the role of PDKs in the development of various metabolic diseases and cancers (Park et al., 2018). Accumulating evidence suggests that alterations in PDKs are associated with the development of several neurological disorders (Jha et al., 2012).
Mammals exhibit four PDK isoforms with different tissue expression levels and functions that are encoded by independent genes (Park et al., 2018). The
To understand the physiological functions of PDK, we analyzed ATP levels, which indicate mitochondrial function, and mtDNA content, which allowed us to quantify the number of mitochondria in the indirect flight muscles of the
Sensitivity to oxidative stress is closely related to neurodegenerative diseases, especially PD. ROS sensitivity is markedly increased in flies lacking
PDKs phosphorylate and inhibit PDH, while PDPs catalyze the reverse reaction, leading to PDH activation. There are two PDP isoforms (PDP1 and PDP2) in mammals (Park et al., 2018), whereas only one PDP isoform exists in
Based on its protective role against oxidative stress, we investigated PDK expression levels under oxidative stress. Surprisingly, H2O2 treatment increased PDK mRNA expression levels in wild-type flies but not in
Owing to recent correlations between mitochondrial metabolism and cancer, small molecules that can modulate PDH activity have been developed. Zachar et al. (2011) developed CPI-613, a lipoate analog that resembles PDH catalytic intermediates and can inactivate PDH via PDK activation. As expected, CPI-613 induced PDK-dependent phosphorylation of PDH and subsequently inhibited PDC activity (Zachar et al., 2011). Thus, we investigated whether CPI-613 administration rescued oxidative stress-induced defects in
In this study, we isolated and characterized the
Consistent with our results, PDCs and their regulators are critically involved in various neurological disorders. PDC deficiency in fetus causes congenital lactic acidosis and CNS malfunction, which ranges from minimal motor impairment to profound intellectual disability with motor deficits (Pliss et al., 2016). A missense mutation in
According to previous studies, the expression of PDK isoforms is regulated by various stress factors, such as starvation, hypoxia, and oxidative stress (Jha et al., 2012). Interestingly, H2O2 treatment induced
Our genetic data suggest that PDC and its regulators may be putative molecular targets for the treatment of
This work was supported by grants from the National Research Foundation of Korea, funded by the Ministry of Science and ICT (grant Nos. 2020R1F1A105165912 and 2016R1A5A2007009) and a Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (grant No. 2021R1A6C101A425).
Y.L., J.K., H.K. (Hyunjin Kim), and J.E.H. designed and performed experiments. Y.L., J.K., and H.K. (Hyongjong Koh) wrote the manuscript. S.K. and K.K. analyzed the data. D.K., J.M.K., and H.K. (Hyongjong Koh) supervised this study.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2022; 45(7): 454-464
Published online July 31, 2022 https://doi.org/10.14348/molcells.2022.5002
Copyright © The Korean Society for Molecular and Cellular Biology.
Yoonjeong Lee1,2,6 , Jaehyeon Kim1,3,6
, Hyunjin Kim1,2
, Ji Eun Han1,3
, Sohee Kim1,3
, Kyong-hwa Kang1,4
, Donghoon Kim1,2,3,4
, Jong-Min Kim5
, and Hyongjong Koh1,2,3,4,*
1Department of Pharmacology, Dong-A University College of Medicine, Busan 49201, Korea, 2Peripheral Neuropathy Research Center, Dong-A University College of Medicine, Busan 49201, Korea, 3Department of Translational Biomedical Sciences, Dong-A University College of Medicine, Busan 49201, Korea, 4Neuroscience Translational Research Solution Center, Dong-A University College of Medicine, Busan 49201, Korea, 5Department of Anatomy and Cell Biology, Dong-A University College of Medicine, Busan 49201, Korea, 6These authors contributed equally to this work.
Correspondence to:hjkoh@dau.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
DJ-1 is one of the causative genes of early-onset familial Parkinson’s disease (PD). As a result, DJ-1 influences the pathogenesis of sporadic PD. DJ-1 has various physiological functions that converge to control the levels of intracellular reactive oxygen species (ROS). Based on genetic analyses that sought to investigate novel antioxidant DJ-1 downstream genes, pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to increase survival rates and decrease dopaminergic (DA) neuron loss in DJ-1 mutant flies under oxidative stress. PDK phosphorylates and inhibits the PDH complex (PDC), subsequently downregulating glucose metabolism in the mitochondria, which is a major source of intracellular ROS. A loss-of-function mutation in PDK was not found to have a significant effect on fly development and reproduction, but severely ameliorated oxidative stress resistance. Thus, PDK plays a critical role in the protection against oxidative stress. Loss of PDH phosphatase (PDP), which dephosphorylates and activates PDH, was also shown to protect DJ-1 mutants from oxidative stress, ultimately supporting our findings. Further genetic analyses suggested that DJ-1 controls PDK expression through hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator of the adaptive response to hypoxia and oxidative stress. Furthermore, CPI-613, an inhibitor of PDH, protected DJ-1 null flies from oxidative stress, suggesting that the genetic and pharmacological inhibition of PDH may be a novel treatment strategy for PD associated with DJ-1 dysfunction.
Keywords: DJ-1, Drosophila, oxidative stress, Parkinson’s disease, pyruvate dehydrogenase kinase
Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by typical motor symptoms and selective loss of dopaminergic (DA) neurons in the substantia nigra (Lang and Lozano, 1998). Various studies have supported the link between DA neuron loss and oxidative stress originating from excess generation or insufficient elimination of reactive oxygen species (ROS) (Henchcliffe and Beal, 2008). In postmortem brain analyses, oxidative damage to macromolecules, such as DNA, proteins, and lipids, has been shown to be substantially elevated in the substantia nigra of PD patients (Bender et al., 2006; Henchcliffe and Beal, 2008). Various PD animal models have been generated using oxidative stress-inducing agents, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat, and 6-hydroxydopamine (6-OHDA) (Betarbet et al., 2000; Brooks et al., 1999; Langston et al., 1983; Ungerstedt and Arbuthnott, 1970). However, the molecular mechanisms underlying oxidative stress-induced degeneration of DA neurons have not been fully elucidated.
PD mainly develops sporadically, but can also occur due to monogenic mutations (Lesage and Brice, 2009). Among the PD-linked genes, DJ-1 is most closely associated with oxidative stress (Henchcliffe and Beal, 2008). Bonifati et al. (2003) reported that DJ-1 is associated with the development of an autosomal recessive early-onset type of familial PD. Further, subsequent studies revealed that DJ-1 plays various roles in cell biology, such as transcriptional regulation, anti-apoptotic signaling, proteostasis, and mitochondrial regulation in response to oxidative stress (Ariga et al., 2013). DJ-1 is sequentially oxidized at its cysteine residues under oxidative stress, and its activity and subcellular localization are regulated by its oxidative status (Ariga et al., 2013). Notably, excessive oxidation leads to DJ-1 inactivation, and this excessively oxidized form has been identified in the brain of patients with sporadic PD (Bandopadhyay et al., 2004; Choi et al., 2006). Such findings suggest that DJ-1 plays a role in the pathogenesis of both sporadic and familial PD.
In this study, the overexpression of pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to enhance survival rates and ameliorate DA neuron loss in
All flies were grown as described previously (Jang et al., 2020). The
For the oxidative stress assays, three or four groups of 30 3-day-old flies (n = 90 or 120) were starved for 6 h and transferred to a vial containing a gel consisting of phosphate-buffered saline (PBS), 5% sucrose, and an oxidative stress agent (5 mM rotenone or 1% H2O2). To determine the role of
For mitochondrial DNA (mtDNA) polymerase chain reaction (PCR), total DNA was extracted from the thoraces of five 3- and 10-day-old male flies. Quantitative real-time (RT) PCR was performed as described previously (Yang et al., 2017). Rp49 genomic DNA levels were used as an internal control. The results are expressed as fold-changes relative to the control. For the ATP assay, the thoraces of five 3-day-old male flies were dissected, and ATP concentration was measured as previously described (Yang et al., 2017). The relative ATP level was calculated as the ratio of ATP concentration to the total protein concentration. The total protein concentration was determined using the bicinchoninic acid assay (Sigma, USA). The results of the mtDNA PCR and ATP assays are expressed as mean ± SD of three independent experiments.
To investigate the change in the number of DA neurons in
To measure the transactivation activity of Sima on the
Total RNA was extracted from five 3-day-old flies and reverse-transcribed, as previously described (Park et al., 2006). Quantitative RT-PCR was performed using SYBR Premix Ex Taq (Takara, Japan) on QuantStudio 3 (Thermo Fisher Scientific, USA) at the Neuroscience Translational Research Solution Center. Rp49 levels were used as internal controls. The results are expressed as fold-changes relative to the control. The average mRNA levels ± SD were obtained from three independent experiments. The following primer pairs were used: rp49 F (GCT TCA AGA TGA CCA TCC GCC C) and R (GGT GCG CTT GTT CGA TCC GTA AC); PDK F (CTG TTT CCA GTC CGA TTC TCA G) and R (CAG AAG GGC GAT CTC CTT CAT G); prel F (CAC CAG GAA CCT GGG AAT GAC) and R (CAG CAC GTA GTT GAA TCC ATT GGA G); PDP F (CTC CAT ATA GAT GTT CAA GTT CGT G) and R (GTA AAC GAA CTC GTT CTC CCT CAG); and Rab39 F (GAC AGC ACC GTG GGC AAG AG) and R (GAC TTG GTG ATC GAA CGG AAG C).
To quantify DA neurons, four major DA neuron clusters from more than 15 brains of each genotype were observed in a blinded fashion to eliminate bias (n = 30-40). One-way ANOVA with Sidak correction was used to compare three or more groups, while Student’s two-tailed
The central nervous system (CNS) receives most of its energy from oxygen-dependent glucose metabolism. Further, mitochondrial PDC mediates a major step in glucose oxidation, namely the irreversible decarboxylation of pyruvate to acetyl-CoA (Jha et al., 2012). PDC activity is tightly regulated by PDK- and PDP-induced phosphorylation of the PDH E1 subunit, which is the first component of PDC (Park et al., 2018). Recent studies have revealed the role of PDKs in the development of various metabolic diseases and cancers (Park et al., 2018). Accumulating evidence suggests that alterations in PDKs are associated with the development of several neurological disorders (Jha et al., 2012).
Mammals exhibit four PDK isoforms with different tissue expression levels and functions that are encoded by independent genes (Park et al., 2018). The
To understand the physiological functions of PDK, we analyzed ATP levels, which indicate mitochondrial function, and mtDNA content, which allowed us to quantify the number of mitochondria in the indirect flight muscles of the
Sensitivity to oxidative stress is closely related to neurodegenerative diseases, especially PD. ROS sensitivity is markedly increased in flies lacking
PDKs phosphorylate and inhibit PDH, while PDPs catalyze the reverse reaction, leading to PDH activation. There are two PDP isoforms (PDP1 and PDP2) in mammals (Park et al., 2018), whereas only one PDP isoform exists in
Based on its protective role against oxidative stress, we investigated PDK expression levels under oxidative stress. Surprisingly, H2O2 treatment increased PDK mRNA expression levels in wild-type flies but not in
Owing to recent correlations between mitochondrial metabolism and cancer, small molecules that can modulate PDH activity have been developed. Zachar et al. (2011) developed CPI-613, a lipoate analog that resembles PDH catalytic intermediates and can inactivate PDH via PDK activation. As expected, CPI-613 induced PDK-dependent phosphorylation of PDH and subsequently inhibited PDC activity (Zachar et al., 2011). Thus, we investigated whether CPI-613 administration rescued oxidative stress-induced defects in
In this study, we isolated and characterized the
Consistent with our results, PDCs and their regulators are critically involved in various neurological disorders. PDC deficiency in fetus causes congenital lactic acidosis and CNS malfunction, which ranges from minimal motor impairment to profound intellectual disability with motor deficits (Pliss et al., 2016). A missense mutation in
According to previous studies, the expression of PDK isoforms is regulated by various stress factors, such as starvation, hypoxia, and oxidative stress (Jha et al., 2012). Interestingly, H2O2 treatment induced
Our genetic data suggest that PDC and its regulators may be putative molecular targets for the treatment of
This work was supported by grants from the National Research Foundation of Korea, funded by the Ministry of Science and ICT (grant Nos. 2020R1F1A105165912 and 2016R1A5A2007009) and a Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (grant No. 2021R1A6C101A425).
Y.L., J.K., H.K. (Hyunjin Kim), and J.E.H. designed and performed experiments. Y.L., J.K., and H.K. (Hyongjong Koh) wrote the manuscript. S.K. and K.K. analyzed the data. D.K., J.M.K., and H.K. (Hyongjong Koh) supervised this study.
The authors have no potential conflicts of interest to disclose.
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