Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's

Glutathione depletion resulting in selective mitochondrial complexI inhibition in dopaminergic cells is via an NO-mediated pathwaynot involving peroxynitrite: implications for Parkinson’s disease

Michael Hsu,* Bharath Srinivas,� Jyothi Kumar,� Rajagopalan Subramanian� and Julie Andersen�

*Department of Molecular Biology, University of Southern California, Los Angeles, California, USA

�Buck Institute, Novato, California, USA

Abstract

An early biochemical change in the Parkinsonian substantia

nigra (SN) is reduction in total glutathione (GSH + GSSG)

levels in affected dopaminergic neurons prior to depletion in

mitochondrial complex I activity, dopamine loss, and cell

death. We have demonstrated using dopaminergic PC12 cell

lines genetically engineered to inducibly down-regulate

glutathione synthesis that total glutathione depletion in these

cells results in selective complex I inhibition via a reversible

thiol oxidation event. Here, we demonstrate that inhibition of

complex I may occur either by direct nitric oxide (NO) but not

peroxinitrite-mediated inhibition of complex I or through H2O2-

mediated inhibition of the tricarboxylic acid (TCA) cycle

enzyme a-ketoglutarate dehydrogenase (KGDH) which

supplies NADH as substrate to the complex; activity of both

enzymes are reduced in PD. While glutathione depletion

causes a reduction in spare KGDH enzymatic capacity, it

produces a complete collapse of complex I reserves and

significant effects on mitochondrial function. Our data suggest

that NO is likely the primary agent involved in preferential

complex I inhibition following acute glutathione depletion in

dopaminergic cells. This may have major implications in terms

of understanding mechanisms of dopamine cell death asso-

ciated with PD especially as they relate to complex I inhibition.

Keywords: complex I, glutathione, Parkinson’s disease, PC12,

reactive oxygen species, reactive nitrogen species.

J. Neurochem. (2005) 92, 1091–1103.

Parkinson’s disease (PD) is an age-related neurodegenerativedisorder which results in the preferential loss of dopamin-ergic neurons of the substantia nigra (SN) pars compacta(Bains and Shaw 1997). Loss of these specific neuronsproduces symptoms which include rigidity, tremors, brad-ykinesia, and loss of motor control (Lang and Lozano 1998).Genetic factors are known to cause PD in rare cases (Gwinn-Hardy 2002), however, environmental toxins coupled withgenetic susceptibility have been implicated in the morecommon sporadic form of the disorder (Siderowf and Stern2003). Although various factors have been correlated withthe disease, the specific molecular mechanisms by whichthey interact to contribute to the disease state are unclear(Lang and Lozano 1998).

Selective inhibition of activity of mitochondrial complex I(NADH:ubiquinone oxireductase) of the respiratory chain isone factor implicated in PD. Activity has been reported to be

Received July 21, 2004; revised manuscript received October 13, 2004;accepted October 14, 2004.

Address correspondence and reprint requests to Julie K. Andersen, 8001Redwood Blvd, Novato, CA 94945, USA.E-mail: [email protected] used: BSO, buthionine sulfoxamine; CI, complex I;

DAF-FM, diacetate, 4-amino-5-methylamino-2¢,7¢-difluorofluoresceindiacetate; DB, decylubiquinone; DCPIP, 2,6-dichlorophenolinodophe-nol; DEA/NO, diethylamine/nitric oxide complex sodium; DMEM,Dulbecco’s modified Eagle medium; dox, doxycycline; DTT, dithio-threitol; Fe-S, iron sulfur; FeTPPS, 5,10,15,20-tetrakis-[4-sulfonatophe-nyl]-porphyrinato-iron[III]; GCL, glutamylcysteine ligase; GSH,glutathione; GSHPx, glutathione peroxidase; GSNO, nitrosoglutathione;GST, glutathione-S-transferase; NGF, nerve growth factor; HBSS,Hank’s balanced salt solution; 4-HNE, 4-hydroxynonenal; H2O2,hydrogen peroxide; KGDH, a-ketoglutarate dehydrogenase; MES, 2-morpholinoethanesulfonic acid; MnTBAP, Mn(III)tetrakis(4-Benzoicacid)porphyrin; NAC, L-N-acetyl cysteine; 7-NI, 7-nitroindazole; NO,nitric oxide; NO+, nitrosonium; N2O3, dinitrogen trioxide; 3-NT, 3-ni-trotyrosine; PD, Parkinson’s disease; S-benz, S-benzylisothiourea; PTIO,2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; sin-1, 3-mor-pholinosyndonimine; SDS–PAGE, sodium dodecyl sulfate polyacryla-mide gel electrophoresis; SN, substantia nigra; SNAP, (+/–)-S-Nitroso-N-acetylpenicillamine; SOD, superoxide dismutase; TCA, tricarboxylicacid; TMPD, N,N,N,N-tetra methyl-p-phenylenediamine.

Journal of Neurochemistry, 2005, 92, 1091–1103 doi:10.1111/j.1471-4159.2004.02929.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 1091–1103 1091

significantly reduced in post-mortem PD SN versus age-matched controls (Schapira et al. 1990) and administrationof known complex I-specific inhibitors [rotenone and1-methyl-4-phenyl 1,2,3,6-tetraphydropyridine (MPTP)] torodents has been demonstrated to mimic many aspects ofdisease pathology (Heikkila et al. 1984; Betarbet et al.2000).

PD is also characterized by decreases in SN levels of totalglutathione (GSH + GSSG); this depletion occurs withindopaminergic neurons in this brain region (e.g. Perry andYong 1986; Sofic et al. 1992; Sian et al. 1994a, 1994b;Pearce et al. 1997). Although glutathione is not the onlyantioxidant molecule reported to be altered in PD, it is one ofthe earliest known biochemical indicator of nigral degener-ation and the magnitude of its depletion parallels the severityof the disease preceding measurable losses in either mitoch-ondrial complex I activity or striatal dopamine content (Perryand Yong 1986; Sian et al. 1994a). Furthermore, it is notreduced in other areas of the PD brain or in brain areas otherthan the SN in diseases affecting the basal ganglia such asmultiple system atrophy or progressive supranuclear palsy(Perry and Yong 1986; Fitzmaurice et al. 2003). Earlyreductions in total SN glutathione levels have also beendetected in animal models of the disease (Kaur et al. 2003).Intravenous delivery of glutathione to a small group ofuntreated PD patients over the period of a month has beenreported to result in significant improvement of disability,suggesting that early glutathione depletion may be atherapeutic target for the disease (Perry and Yong 1986;Sechi et al. 1996; Fitzmaurice et al. 2003).

GSH plays an important role in the adult brain byremoving oxidants formed during normal cellular metabo-lism such as oxygen utilization by the mitochondria; itsdecreased availability in the brain has been demonstrated tolead to mitochondrial damage (Jain et al. 1991). Previouslypublished studies from our own laboratory demonstrated thattotal glutathione reduction within dopaminergic PC12 cellsas a model of the disease leads to selective complex Iinhibition via an unidentified thiol-dependent oxidation eventwhich is reversible by the thiol-reducing agent, dithiothreitol(DTT; Jha et al. 2000). GSH normally acts through acombination of various reduction and conjugation reactionsto protect cells against both exogenous toxicants and thereaction of endogenous compounds (Krzywanski et al.2004). It acts, for example, in concert with glutathioneperoxidase (GSHPx; EC 1.11.1.9) as a substrate for thereduction of hydrogen peroxide (H2O2) and lipid peroxidesand for removal of peroxynitrite formed from the interactionof superoxide with nitric oxide (NO; van der Vliet et al.1998). GSH can also directly conjugate with highly reactiveelectrophilic compounds either non-enzymatically or viaactivity of the glutathione-S-transferases (GST; EC 2.5.1.18;Meister and Anderson 1983; Townsend and Tew 2003). GSHconjugates, for example, with NO to form nitrosoglutathione

(GSNO) as well as with the lipid peroxidation by-product4-hydroxynonenal (4-HNE) (Dickinson and Forman 2002a,2002b). Decreases in GSH levels could presumably result inan increase in levels of all of these oxidant species.Interestingly, several of these agents have either beenimplicated in PD itself or found to affect the activity ofmitochondrial enzymes known to be altered in the disorder.For example, both 4-HNE protein conjugates and nitrated ornitrosylated modifications to proteins linked to the diseaseincluding a-synuclein, tyrosine hydroxylase (EC 1.14.16.2),and Parkin have been reported in the PD brain and/or inanimal models of the disease (Yoritaka et al. 1997; Ara et al.1998; Good et al. 1998; Przedborski and Jackson-Lewis1998; Kuhn et al. 1999; Giasson et al. 2000; Chung et al.2004). Treatment of isolated rodent brain mitochondria withpathological levels of H2O2 has been found to result ininhibition of KGDH activity, a mitochondrial enzyme whoselevels have been reported to be selectively decreased in PD(Tretter and Adam-Vizi 2000). In this study, we haveidentified possible critical events involved in glutathionedepletion-mediated complex I inhibition which occur viaseparate NO and H2O2-mediated pathways, the relativecontribution of each to this process and how they may impacton subsequent mitochondrial function.

Experimental procedures

Materials

Chemicals used for all assays were obtained from Sigma (St Louis,

MO, USA) unless otherwise noted. Absorbance readings in the UV/

visible range were measured using a SpectraMax 340PC spectro-

photometric microplate reader (Molecular Devices, Palo Alto, CA,

USA) and fluorescent assays using a SpectraMax Gemini fluores-

cent microplate reader (Molecular Devices).

Creation of doxycycline inducible anti-c-GCL PC12 cell lines

Permanent anti-c-glutamyl cysteine ligase (GCL) PC12 cell lines

were produced as described previously, except Lipofectamine 2000

(Invitrogen, Carlsbad, CA, USA) was used for all cell transfections

(Jha et al. 2000).

Growth and differentiation of anti-c-GCL PC12 cells

Cells were grown on poly-L-lysine coated plates (Greiner, Long-

wood, FL, USA) in medium containing 1 · Dulbecco’s modified

Eagle medium (DMEM; Cellgro, Herndon, VA, USA), 10% horse

serum, 5% tetracycline (tet) system-approved fetal bovine serum

(Clontech, Palo Alto, CA, USA), 200 lg/mL geneticin (G418),

200 lg/mL hygromycin B, and 10 mL/L of antibiotic antimycotic

solution (Cellgro). For all subsequent experiments, cells were pre-

treated with 50 ng/mL nerve growth factor (NGF) 7S for 2 days to

promote differentiation. Doxycycline (dox) was then added to the

medium at 20–40 lg/mL for 24 h to induce down-regulation of total

glutathione levels. For dox removal experiments, cells were washed

with Hank’s balanced salt solution (HBSS) and grown for 24 h in

dox-free media. Samples for catalase treatment were incubated with

1092 M. Hsu et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 1091–1103

1 mg/mL catalase for 12 h and then fresh media were added which

included 40 lg/mL dox and 1 mg/mL catalase followed by 24 h

incubation. For 7-nitroindazole (7-NI) or S-benzylisothiourea

(S-benz) treatment, cells were incubated with 1 mM of the agent

for 24 h while being treated simultaneously with 40 mg/ll dox.

(±)-S-Nitroso-N-acetylpenicillamine (SNAP) was added at 100 mM

for 5 h. 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide

(PTIO) was added at 2 (M for 2 h then with either SNAP or dox.

3-Morpholinosydnonimine (sin-1) was added at a 1 mM concentra-

tion for 10 min directly into the culture medium prior to cell harvest.

5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (FeT-

PPS, Calbiochem, San Diego, CA, USA) was added during a 12 h

pre-treatment (100 mM) followed by a 24 h co-treatment with either

40 lg/mL dox or sin-1. Mn(III)tetrakis(4-Benzoic acid)porphyrin

(MnTBAP) was added at 100 mM concentration along with

40 mg/mL dox during a 24 h co-treatment.

c-GCL activity

Cell homogenates resuspended in 100 mM Tris–Cl, pH 8.0 were

used to assay c-GCL activity levels. Absorbance of 340 nm was

measured for 3 min at 37�C on a microplate reader as previously

described (Seelig and Meister 1985). Values for this and all

subsequent assays were normalized per protein using the Bio-Rad

reagent (Bio-Rad Laboratories, Hercules, CA, USA).

Glutathione levels

Total glutathione levels were measured using the Bioxytech GSH/

GSSG 412 assay kit (Oxis Research, Portland, OR, USA) per the

manufacturer’s instructions.

Hydrogen peroxide assay

Hydrogen peroxide levels were measured in cell media using the

Amplex Red assay kit (Molecular Probes, Eugene, OR, USA).

Various dilutions from 1 to 100 lg of sample proteins were assayed

per the manufacturer’s instructions (Zhou et al. 1997). Fluorescencewas measured at an excitation wavelength of 540 nm and an

emission wavelength of 590 nm. Samples below fluorescent

saturation were used for data analysis.

4-Hydroxynonenal assay

4-hydroxynonenal (4-HNE) levels where measured by use of the

Bioxytech LPO-586 kit (Oxis Research) per the manufacturer’s

instructions.

Nitric oxide assay

Nitrosonium (NO+) levels were measured using the fluorescent

probe 4-amino-5-methylamino-2¢,7¢-difluorofluorescein diacetate

(DAF-FM diacetate) from Molecular Probes. Cells where grown

on glass-chambered slides (Nalge ne) coated with poly-L-lysine,

treated with NGF, then either in the absence or presence of dox ±

7-NI as described above. After treatment, DAF-FM diacetate was

loaded directly into the medium at 10 mM for 1 h. After loading,

the medium was changed and fluorescence scanned on a Nikon

PCM-2000 laser confocal scanning microscope (Nikon Melville,

NY, USA) at excitation and emission wavelengths of 495 nm and

515 nm, respectively. Images were captured using a Nikon DXM

1200 digital camera. Fluorescence quantitation was performed

using Bitplane advanced imaging software.

Mitochondrial preparation

Cells were washed and resuspended in ice-cold isolation buffer

(320 mM sucrose, 5 mM Tes, and 1 mM EGTA, pH 7.4) followed by

homogenization in a Dounce homogenizer. Homogenates were

centrifuged at 1000 g for 5 min at 4�C. Supernatant containing the

mitochondria was saved and pellet resuspended in isolation buffer

and rehomogenized. Centrifugation of the resuspended homogenate

was repeated and the supernatants pooled. The pooled supernatant

was centrifuged at 8000 g for 10 min at 4�C. The pellet containing

the mitochondria was resuspended in 50 lL isolation buffer for

mitochondrial complexes I, II (EC 1.10.2.2), and IV (EC 1.9.3.1),

KGDH, and citrate synthase (EC 2.3.3.1) enzyme activity assays.

Complex I activity

Activity was assayed in homogenates from isolated mitochondria as

rotenone-sensitive NADH dehydrogenase activity by measuring

DCPIP (2,6-dichlorophenolindophenol) reduction in mitochondrial

extract following addition of 200 lM NADH, 200 lM decylubiqui-

none (DB), 2 mM KCN, and 0.002% DCPIP in the presence and

absence of 2 lM rotenone (Trounce et al. 1996). The reaction was

performed at 600 nm at 30�C.

Complex II–III activity

Activity was assayed on isolated mitochondria preparations.

Mitochondrial samples were added to buffer containing 50 mM

Tris–SO4 (pH 7.4), 100 mM EDTA, 2 mM succinate, and 1%

maltoside. The assay was initiated with 100 mM cytochrome c and

absorbance was measured at 550 nm at 30�C in the presence and

absence of 4 mM antimycin A.

Complex IV activity

Activity was assayed on isolated mitochondria preparations.

Mitochondrial samples were added to buffer containing 10 mM

KPO4 (pH 7.4), 100 mM KCl, 0.025% maltoside, and 80 mM ferro-

cytochrome c; addition of mitochondrial sample was used to initiate

the reaction. Absorbance was measured at 550 nm at 30�C.

a-Ketoglutarate dehydrogenase activity

KGDH activity was assayed as the rate of NAD+ reduction at

340 nm upon addition of 5.0 mM MgCl2, 40.0 lM rotenone, 2.5 mM

a-ketoglutarate, 1.0 mM CoA, 0.2 mM thymine pyrophosphate, and

1.0 mM NAD+ (Nulton-Persson and Szweda 2001).

Citrate synthase activity

One microgram of mitochondrial preparation was added to a

reaction mixture consisting of 0.1 M Tris–HCl (pH 8.0), 0.3 mM

acetyl-CoA, and 0.1 mM 5,5¢-dithiobis(2-nitrobenzoic acid). The

assay was initiated by addition of a final concentration of 0.5 mM

oxaloacetate. Activity was measured in a total volume of 200 lL at

30�C at 412 nm.

Oxygen consumption

Mitochondrial respiration was assayed in digitonin-permeabilized

cells (2 lg/mL) in a buffer containing 125 mM KCl, 2 mM KH2PO4,

1 mM MgCl2 and 20 mM HEPES pH 7.0 (Murphy et al. 1996) at37�C using a Clarke electrode (Hansatech, Kings Lynn, UK).

Respiration was calculated as the rate of oxygen consumption using

either 5.0 mM a-ketoglutarate/5.0 mM malate as substrates for

Glutathione affects mitochondrial complex I via NO not ONOO 1093


KGDH or 5.0 mM glutamate/5.0 mM malate as substrates for

complex I in presence or absence of selective inhibition with

0–100 nM arsenite (KGDH) or 0–2.0 lM rotenone (complex I),

respectively. Then, 5.0 mM ascorbate/10 lM TMPD (N,N,N,N-tetra

methyl-p-phenylenediamine) were used to assay KCN-inhibitable

(1 mM) cytochrome c oxidase (complex IV)-driven respiration as a

negative control. FCCP (10 lM) was added as uncoupler to assess

maximum respiration rates.

ATP/ADP ratio

The ApoGlow Rapid Apoptosis Screening Kit (Cambrex, East

Rutherford, NJ, USA) was used to measure the ATP/ADP ratio

based on the luciferase assay according to the manufacturer’s

instructions. Relative luminescent units were measured on a Turner

T-20E luminometer (Turner, Sunnyvale, CA, USA).

Analysis of presence of nitrosated mitochondrial proteins

Biotin switch modification of mitochondrial proteins isolated from

treated versus untreated cells was performed as previously

described (Chung et al. 2004); this involves alkylation of reduced

–SH groups, selective –NO reduction to –SH, and addition of biotin

adduct (biotin-HPDP) to label –SH groups. Isolated fractions

containing biotinylated (formerly nitrosated) proteins were separ-

ated on 10–20% pre-cast SDS–PAGE (sodium dodecyl sulfate

polyacrylamide gel electrophoresis) in an X-Cell Sure Lock Mini

Cell Apparatus (Invitrogen) with a SDS running buffer [50 mM

2-morpholinoethanesulfonic acid (MES), 50 mM Tris base, 0.1%

SDS, 1 mM EDTA] at 100 V for 1.5–2 h. Proteins were transferred

onto nitrocellulose membrane and probed with antibiotin polyclonal

antibody (Molecular Probes). Duplicate mitochondrial protein

samples were subjected to strep-avidin purification followed by

SDS–PAGE gel seperation as previously described (Chung et al.2004), fixation with 10% methanol, 7% acetic acid for 30 min, and

subsequently staining with Sypro Ruby followed by destaining in

10% methanol/7% acetic acid.

Statistical analysis

Data are expressed as mean ± SD and significance testing was

performed using ANOVA.

Results

Dox-dependent down-regulation of GSH synthesis results

in increased levels of H2O2, 4-HNE, and NO+

GSH is synthesized in the cytosol and transported into themitochondria by an energy-dependent mechanism (Meister1988). The rate-limiting enzyme in its synthesis is theenzyme (– glutamyl cysteine ligase (GCL) (EC 6.3.2.2)(Seelig and Meister 1985). We have previously reported theability to reduce cellular and mitochondrial levels of totalglutathione within dopaminergic PC12 cells via reducingactivity of the catalytic subunit of GCL via its antisenseexpression (Jha et al. 2000; Figs 1a and b). The use of senseGCL and empty vector controls in these earlier studiesverified that the CI inhibition is specific to anti-GCLexpression (Jurma et al. 1998; Jha et al. 2000). Depletion

in total glutathione to similar levels have been reported tooccur in the Parkinsonian brain (e.g. Perry and Yong 1986;Sofic et al. 1992; Sian et al. 1994a, 1994b); this decreaseoccurs within dopaminergic SN neurons (Pearce et al. 1997).The decrease in both GCL activity and glutathione levels arereversible upon dox removal from the media.

Reduction in total glutathione levels resulted in an increasein the cellular oxidant species H2O2, 4-HNE, and NO +(Figs 2a–d). Dox-dependent increases in both H2O2 and4-HNE levels are preventable via co-treatment with catalasedemonstrating that both are H2O2-dependent processes.Increases in both H2O2 and NO + were reversible upondox removal from the media.

Total glutathione depletion results in selective inhibition

of complex I which is due to elevation in nitric oxide

species not involving peroxynitrite

We have previously reported that glutathione depletionresults in a selective inhibition of complex I activity similarto that found to occur in the Parkinsonian brain (Fig. 3a;Parker et al. 1989; Schapira et al. 1990; Jha et al. 2000).

Fig. 1 Dox dosage-dependent reduction in GCL activity and total

glutathione levels in dopaminergic anti-GCL PC12 model. (a) GCL

activity following 24 h treatment in 0 (control), 20, or 40 lg/mL dox and

24 h following dox removal (100% GCL activity, 2.8 ± 0.3 nmol/min/

mg of protein). (b) GSH levels following 24 h treatment in 0, 20, or

40 lg/mL dox and 24 h following dox removal (100% GSH value, 20.0

(2.0 nmol/mg protein); *p < 0.01 compared to no dox control;

**p < 0.01 compared to 20 mg/mL dox; ***p < 0.05 compared to no

dox control. Values are reported as percentage of untreated control.

Data represent triplicate values from three separate experiments.

1094 M. Hsu et al.


This inhibition is reversible upon dox removal (Fig. 3a) orDTT addition (Jha et al. 2000). Complex I inhibitionperformed in the presence of excess substrate is insensitiveto catalase and is therefore H2O2-independent (and 4-HNE-independent). It is, however, significantly attenuated by thenNOS inhibitor 7-NI and further attenuated by the inducibleNOS inhibitor S-benz and by the NO scavenger PTIO,suggesting it is NO-mediated. Generation of peroxynitrite, anNO metabolite, via sin-1 resulted in complex I inhibition incontrol cells which was attenuated by the peroxynitritedecomposition agent FeTPPS; however, FeTPPS did notprevent the glutathione depletion-mediated complex I inhi-bition. MnTBAP, a SOD mimetic and subsequent peroxyni-trite scavenger, was also tested in the glutathione-depletedcells and found to have no protective effects in terms ofcomplex I inhibition. Glutathione depletion-mediated inhibi-tion of complex I was, however, prevented by the NOscavenger PTIO as observed in cells treated with theNO+ generator SNAP as a positive control. These datasuggest that glutathione depletion-mediated elevations inlevels of a NO event not involving ONOO are involved in thereversible inhibition of complex I.

Complex II displayed a small (15%) reduction inactivity which is catalase-sensitive and reversible uponremoval of dox; however, no change was noted in

complex IV activity following glutathione depletion(Figs 3b and c). This verifies our previous findingssuggesting that complex I in dopaminergic cells appearsto be preferentially affected by glutathione depletion (Jhaet al. 2000).

The TCA cycle enzyme KGDH is inhibited by H2O2

generated via glutathione depletion

a-KGDH is known to play a key role in limiting NADHsubstrate production during oxidative stress and its levels areinhibited in the PD SN (Tretter and Adam-Vizi 2000). Itsinhibition could therefore also impact on complex I activitylevels. Direct enzymatic assay of a-KGDH following gluta-thione depletion in our cells demonstrates a 40% decrease in itsactivity which is attenuated in the presence of catalase andreversible upon dox removal (Fig. 4a). Activity of citratesynthase, a TCA cycle enzyme known to be unaffected byH2O2 (Kumar et al. 2003), was not significantly altered bydox-induced glutathione reduction.

Effects of glutathione depletion-mediated effects on

complex I versus KGDH substrate-driven mitochondrial

respiration rates

Glutathione depletion results in significant inhibition of bothKGDH and complex I activities but via generation of

(a) (b)

(c) (d)

Fig. 2 Hydrogen peroxide, 4-HNE, and nitrosonium (NO+) levels are

increased following glutathione depletion. (a) Hydrogen peroxide lev-

els as assessed via amplex red fluorescence in cells treated for 24 h in

0 (control), 40 lg/mL dox, 24 h following dox removal or ± catalase.

Values are reported as arbitrary fluorescence units. (b) 4-HNE levels

in cells treated in 0 (– dox) or 40 lg/mL dox (+ dox) ± catalase (+cat).

Values are reported as percentage untreated controls (100% HNE

value, 1.37 ± 0.03 mM). (c) Nitric oxide levels assayed via DAF-FM

fluorescence in cells treated for 24 h in 0 or 40 lg/mL dox and 24 h

after dox removal from the media. (d) Fluorescent images of live anti-

GCL cells loaded with DAF-FM diacetate following treatment with 0

(control), 20, or 40 lg/mL dox for 24 h; *p < 0.01 compared to no dox

control; **p < 0.01 compared to dox-only treatment. Data represent

triplicate values from three separate experiments.



different oxidant species. In order to assess the relativeimportance of inhibition of each via glutathione depletion foroverall mitochondrial function, we assessed the percentageinhibition of each at which complex-specific substrate-drivenoxygen consumptionwas affected. Titration of KGDH activitywas performed in the presence of increasing concentrations ofarsenite and complex I activity with increasing rotenoneconcentrations. Citrate synthase activity used as a normalizingcontrol did not vary significantly between treated and untreatedcell populations (data not shown). These were compared withuncoupled oxygen consumption rates in treated versusuntreated cells using a-ketoglutarate (KG)/malate or glutam-ate/malate as substrates in the presence of increasing concen-trations of arsenite or rotenone. Oxygen consumptions rateswere plotted versus enzyme activities with increasing concen-trations of inhibitor in order to assess the affects of enzymeinhibition on mitochondrial respiration (Figs 5a and b). Inuninduced cells, decreases in oxygen consumption rates onlyreached significance when activity of the KGDH enzyme wasinhibited by approximately 65%, suggesting a large spareKGDH capacity in these cells. In dox-induced cells, theinhibitory effects of arsenite addition onKG/malate uncoupledoxygen consumption rateswas observedwhen enzyme activityhad been reduced to only 35%, demonstrating a reduction butnot complete loss of the reserve KGDH capacity. For complexI, inhibiting activity by as little as 17% resulted in decreasedcomplex I substrate-driven respiration in NGF-differentiateduntreated cells and glutathione depletion results in an imme-diate loss in respiration at the smallest concentrations ofrotenone used (5% inhibition). This suggests that inhibition ofcomplex I is rate-limiting in regards to mitochondrial functionversus that of a-KGDH.

Glutathione depletion results in a decrease in ATP/ADP

ratios

A decrease in the ATP/ADP ratio was also observed in dox-treated cells which was reversible upon removal of dox from

Fig. 3 Mitochondrial complex I, II–III, and IV activities in glutathione-

depleted cells. (a) Complex I activity in cells treated for 24 h in 0

(–dox), 40 lg/mL dox (+dox) or 24 h following dox removal or ± cat-

alase (+cat), 7-NI ± S-Benz, FeTPPS, PTIO, or MnTBAP (100% va-

lue, 300 ± 15 nmol/min/mg of mitochondrial protein). Control cells

treated with either Sin-1 ± FeTPPS or SNAP ± PTIO were used as

negative and positive controls, respectively. (b) Complex II–III activity

in cells treated for 24 h in 0, 40 lg/mL dox, 24 h following dox removal

or ± catalase (100% value, 12.8 ± 0.73 nmol/min/mg of mitochondrial

protein). (c) Complex IV activity in cells treated for 24 h in 0 or 40 lg/

mL dox (100% value, 160.4 ± 22.3 nmol/min/mg). *p < 0.01 com-

pared to no dox control; ^p >0.1 compared to dox-only treatment;

**p < 0.01 compared to dox-only treatment. Data represent triplicate

values from three separate experiments.

Fig. 4 KGDH and citrate synthase activities in glutathione-depleted

cells. (a) KGDH activity in cells treated for 24 h in 0 (– dox), 40 lg/mL

dox (+ dox), 24 h following dox removal or ± catalase (cat; 100%

value, 11.51 ± 0.19 nmol/min/mg mitochondrial protein). (b) Citrate

synthase activity in cells treated for 24 h in 0 or 40 lg/mL dox (100%

value, 751 ± 8 nmol/min/mg mitochondrial protein). *p < 0.01 com-

pared to control; ^p >0.05 compared to control. Data represent tripli-

cate values from three separate experiments.

1096 M. Hsu et al.


the media (Fig. 6), suggesting an impact of glutathionedepletion on mitochondrial energy production. Significantcell loss was not observed at the concentration of dox andtime points used for these experiments (data not shown).

Potential nitrosation sites within the subunits of complex I

To investigate possible nitrosation of mitochondrial proteinsincluding complex I following glutathione depletion andelevation of NO+ levels, we performed nitrosation assays inisolated mitochondria from treated cells versus untreatedcontrols. This method (also called the ‘biotin switch’ method)

involves substitution of –NO groups on protein cysteines withbiotin via sequential alkylation of existing protein –SH groupsto prevent their oxidation, selective reduction of existingprotein –NO groups to –SH, and addition of a biotin adductwhich reacts with the newly reduced –SH groups (biotin-HDPD); the presence of biotin-labeled (nitrosated) proteins arethen verified by biotin western blot analysis (Chung et al.2004). Biotinylated proteins are then purified by runningthrough strep-avidin columns, run on SDS–PAGE gel, andstainedwith syproruby.Using thismethod,we observed a largeincrease in the number of nitrosated proteins from the treateddopamine cell mitochondria suggesting that glutathionedepletion can result in widespread protein nitrosation events(Fig. 7).

We next analyzed the subunits of human and rodentcomplex I for the presence of a proposed nitrosation motif asputative targets for the oxidative effects of total glutathionedepletion in our cell model (Table 1). A total of 43 cysteineswithin 22 subunits of rodent complex I and 35 cysteineswithin 16 subunits of human complex I were found to bewithin sequences adhering to various degrees to the putativenitrosation site. The cysteines within sequences following thehighest degree of adherence to the motif for both rodent andhuman were found within the Ndufa9 and Ndufs2 subunits.Cysteines within both rodent and human complex I subunitsfound within tetrapeptide sequences implicated in iron–sulfurcluster formation were also identified and these werecompared with those found within putative nitrosation motifsto look for overlap between the two (Table 2). The Fe-Scluster cysteine found within the most conserved nitrosationsite in both rodent and human was one found within complexI subunit Ndufs1.

Discussion

In an earlier publication using the described anti-GCL cellsystem to assess the affects of total glutathione depletion onmitochondrial function in dopaminergic cells as a model forParkinson’s disease, we demonstrated that total glutathionedecreases resulted in selective inhibition of complex I activity

Fig. 5 Effect of dox treatment on KGDH and complex I respiration

thresholds. Effects of dox induction on inhibitor-sensitive substrate-

specific respiration as a function of enzyme activity. (a) Uncoupled

KGDH-driven respiration rates using KG/malate as substrates were

measured in induced versus uninduced cells following addition of

increasing concentrations of arsenite. This was plotted versus KGDH

inhibition, taking maximal (100%) inhibition at 100 nM arsenite. (b)

Uncoupled complex-driven respiration rates using pyr/malate as sub-

strates were measured in induced versus uninduced cells following

addition of increasing concentrations of rotenone. This was plotted

versus complex I inhibition, taking maximal (100%) inhibition at 2 lM

rotenone. Values are reported as percentage oxygen consumption

rate or percentage enzyme activity versus cells with no inhibitor

addition. Curves are representative of at least three separate experi-

ments.

Fig. 6 The ATP/ADP ratio is reduced by glutathione depletion. ATP/

ADP ratio in cells treated for 24 h in 0 or 40 mg/mL dox or 24 h fol-

lowing dox removal. *p < 0.01 compared to control. Data represent

triplicate values from three separate experiments.



which was reversible by the thiol-reducing agent, DTT. Ourstudies demonstrated that reductions in total glutathione asobserved in the parkinsonian SN, the area affected in thedisorder, can directly result in selective reduction in mitoch-ondrial complex I activity, another hallmark of the disease (Jhaet al. 2000). However, it did not allow us to identify themechanism by which this occurs or the exact oxidant speciesresponsible (although the fact that it was reversed by DTTsuggested that it was a reversible thiol oxidation event). Inorder to identify the oxidant species involved, we assessedcomplex I activity in glutathione-depleted PC12 cells in theabsence and presence of oxidants which are known to interactwith glutathione species. Catalase (EC 1.11.1.6), which breaksdown H2O2 to water, did not protect against dox-inducedinhibition of complex I. Levels of the lipid peroxidation by-product 4-HNE, another molecule known to interact withglutathione-related species (Siems and Grune 2003), wereincreased following total glutathione depletion; however, thisis unlikely to be the culprit for the observed decrease incomplex I activity as its increase was catalase-sensitive andtherefore also due to elevation in H2O2 levels. Inhibition ofcomplex I was, however, attenuated by the neuronal NOSinhibitor 7-NI and this attenuationwas increased by addition of

the iNOS inhibitor S-benz. Glutathione can normally interactwith NO to form GSNO, which has been speculated to be partof an antioxidant defense mechanism within nigral dopamin-ergic neurons which prevents NO-induced toxicity (Rauhalaet al. 1998). NO can also interact with superoxide to formperoxynitrite which can also be removed by GSNO in aconcentration-dependent manner (Rauhala et al. 1998;Torreilles et al. 1999). While treatment with the peroxynitritegenerator SIN-1 did result in inhibition of complex I in ourcells, whichwas attenuated by the peroxynitrite decompositionagent FeTPPS, in contrast FeTPPS did not prevent dox-inducible inhibition. MnTBAP, a superoxide dismutasemimetic, which prevents the formation of ONOO– viasuperoxide and NO, also does not prevent the dox-inducibleinhibition of complex I. This indicates that peroxynitrite isunlikely to be the NO species responsible for the complex Iinhibition following glutathione depletion. Because peroxy-nitrite is the agent responsible for nitration of protein tyrosineresidues at position 3 of their phenolic rings (3-NT formation;Halliwell 1997), it suggests that 3-NT formation is also notresponsible for complex I inhibition following acute glutathi-one depletion. This is further supported by the fact thatalthough 3-NT is an irreversible adduct (Drew and Leeuwen-burgh 2002), complex I inhibition was found to be reversibleeither when glutathione levels were returned to normal via doxremoval from the media (this study) or after DTTaddition (Jhaet al. 2000). In addition, the effects of acute glutathionedepletion on complex I were reversed by the NO scavengerPTIOwhich also reversed the effects of exposure of cells to theNO+ generator SNAP.

Direct exposure of cells to NO in several in vitro studieshas been reported to result in complex I inhibition butwhether this is a consequence of reversible nitrosation orirreversible nitration events (or both) and the physiologicalrelevance of each has been hotly debated; many studies havebeen performed by bolus addition of either NO donors orONOO (Clementi et al. 1998; Borutaite et al. 2000; Orsiet al. 2000; Riobo et al. 2001; Yamamoto et al. 2002;Murray et al. 2003). Our studies demonstrate that glutathionedepletion-mediated mitochondrial complex I inhibition indopaminergic cells is via a reversible NO-dependent eventinvolving an NO species other than ONOO, either NOradical or its oxidized congener, NO+. NO radical caninteract with labile iron within the several iron–sulfur (Fe-S)centers located within complex I resulting in loss of an iron–sulfur dinitrosyl complex which would lead to irreversibleinhibition of the complex (Granger and Lehninger 1982;Hibbs et al. 1988; Henry et al. 1993; Hentze and Kuhn1996; Ponka and Richardson 1997; Richardson and Ponka1997). As inhibition following glutathione depletion is DTT-reversible, this is therefore not the mechanism by whichinhibitory affects on complex I activity are produced.As complex IV activity was also not impacted duringglutathione depletion, this implies that NO radical is likely

Fig. 7 Increased protein nitrosation following glutathione depletion as

assayed by the biotin switch method. (a) Antibiotin western blot ana-

lysis of mitochondrial samples from treated versus non-treated cells

following biotin switch labeling of protein nitrocysteines. Lane 1, trea-

ted cells; lane 2, untreated control cells; lane 3, control cells without

alkylation; lane 4, control cells without biotin-HPDP addition. (b)

Syproruby staining of mitochondrial samples from treated (lanes 5–7)

versus untreated control cells (lanes 1–3) following strep-avidin col-

umn purification of biotin-labeled proteins. Lanes 1,5 column flow-

through; lanes 2,6 wash; lanes 3,7 column eluate.

1098 M. Hsu et al.


rapidly oxidized to NO+ in the mitochondria which ismembrane-rich rich and where O2 is found in high concen-trations (Brown et al. 1995; Ghafourifar and Richter 1997;Liu et al. 1998; Lopez-Figueroa et al. 2000; Elfering et al.2002). DAF-FM fluorescence, an indicator of NO+ levels,was in fact found to be elevated following glutathionedepletion in our studies. NO+ is considered the ‘quintessen-tial’ S-nitrosation agent reacting with cysteine thiols withinproteins to form reversible nitrosocysteines which can affectprotein function (Gross and Wolin 1995). Nitrosation ofcritical thiols which affect regulation of protein function havebeen previously described for other enzymes includingglyderaldehyde-3-phosphate dehydrogenase (EC 1.2.1.9),caspases, transglutaminase (EC 2.3.2.13), and the ryanodinereceptor (Mohr et al. 1996; Dimmeler et al. 1997; Melinoet al. 1997; Xu et al. 1998). We propose that our data incombination with the known chemistry of NO speciesdemonstrates that formation of NO+ following acute deple-tion of glutathione in our dopaminergic cell model results inreversible S-nitrosation of mitochondrial complex I and itssubsequent enzymatic inhibition. Treatment of isolated ratheart mitochondria or murine macrophage cell lines with NOdonors has previously been shown to result in complex Iinhibition which is reversible by DTT or addition ofmembrane-permeable GSH ethyl ester (GSHEE; Clementiet al. 1998; Borutaite et al. 2000). These data demonstratesthat NO+ elevation such as observed in our cells is capable ofresulting in complex I inhibition by a reversible thiol-dependent oxidation. Increased S-nitrosation of complex Isubunits due to chronic glutathione depletion may representan aberrant pathophysiological event akin to constitutivekinase expression followed by chronic protein phosphoryla-tion in an otherwise reversible signaling pathway. Indeed,inhibition of complex I via S-nitrosation of necessary thiolresidues has been proposed to occur only if the reducingpotential of the cells is impaired such as during glutathionedepletion (Beltran et al. 2000).

Differential reactivity of cysteinemoieties towardsNO+ hasbeen proposed to be a function of both accessibility andnucleophilicity based on the composition of adjacent residues(Stamler et al. 1997; Jaffrey et al. 2001). Analysis of thesubunits of complex I for potential nitrosation sites reveals thattwo cysteines located in the Ndufa9 and Ndufs2 subunits arefound within sequences displaying high adherence to aproposed nitrosation motif. Mutations in the nuclear geneencoding the structural 49 kDa Ndufs2 protein are associatedwith inherited complex I deficiencies suggested to be the resultof compromised complex assembly or stability (Loeffen et al.2001; Ugalde et al. 2004). This subunit has also beendemonstrated to undergo nitrative damage following exposureof isolated bovine heart mitochondria to peroxynitrite whichmay also impact on complex activity (Murray et al. 2003).

Table 1 List of putative S-nitrosylation motifs within different subunits

of mitochondrial complex I (mouse and human)

Subunit Human Score Number Mouse Score Number

Ndufa5 AVCN P 1 AVCD PP 1

Ndufa8 AQCD PP 3 AQCD PP 3

MLCR P MLCR P

RHCR PP RHCR PP

Ndufa9 SVCH P 3 PPCR P 1

PPCR P –

YRCD PPPP YRCD PPPP

Ndufa10 VQCK P 3 VHCK PP 2

TICD PP –

EKCE PPPP EMCE PP

Ndufab1 QLCR P 1 HLCR P 1

Ndufb2 0 RGCR P 1

Ndufb5 0 LSCR P 1

Ndufb LKCK PP 3 LKCK PP 3

LACK P LACK P

DYCE PP DYCE PP

Ndufb8 0 VMCK P 1

Ndufb10 TECK PPP 1 TECK PPP 1

Ndufs1 QACE PP 4 QACE PP 5

GNCR PP GNCR PP

PICD PP PICD PP

GECD PPP GECD PPP

– SFCE PP

Ndufs2 RKCD PPPP 3 RKCD PPPP 3

YLCR P YLCR P

YRCK PP YRCK PP

Ndufs3 – 0 VWCR P 1

Ndufs4 – 0 SVCR P 1

Ndufs5 GRCH PP 2 ARCH PP 2

KECK PP KECK PP

Ndufs6 TFCR P 2 – 1

IACD PP IACD PP

Ndufs7 RGCD PP 1 RGCD PP 1

Ndufs8 IACK P 2 IACK P 2

KLCE PP KLCE PP

Ndufv1 GTCK P 4 GTCK P 4

TICR P TICR P

SVCE PP SVCE PP

TPCR P TPCR P

Ndufv2 FSCE PP 1 FCCE PP 1

Nd4l AACE PP 1 AACE PP 1

B14.7 – TQCH P 1

Number of putative motifs

Motif Arbitratry score Human Mouse

cys-base P 13 14

cys-acid/base-cys-base PP 17 20

base-cys-acid PPP 2 2

polar-base-cys-acid PPPP 3 2



Complex I also consist of at least seven subunits which containsuch Fe-S clusters made up of approximately 39 cysteineresidues, all of which could be impacted by glutathionedepletion to various degrees. A cysteine within the 75 kDaNdufs1 subunit situated within one of the complex’s iron–sulfur centers is within a fairly conserved nitrosation site.Interestingly, this subunit was previously identified as a

potential site for reversible glutathionylation following GSHoxidation (Taylor et al. 2003). Mutations within this subunitare also linked to inherited complex I deficiencies which areassociated with ataxia, hypotonia, and psychomotor retarda-tion (Benit et al. 2001).

Besides complex I, levels of the TCA cycle enzymeKGDH have also be demonstrated to be inhibited in PD(Mizuno et al. 1994). This could indirectly act on complex Iby limiting NADH as substrate for the complex. KGDHactivity is inhibited following glutathione depletion in aH2O2-dependent mechanism in our cells resulting in adecrease in the spare KGDH enzymatic capacity necessaryfor mitochondrial function to about 35%. However, inhibi-tion of complex I in the presence of excess substrate by aslittle as 5% in our cells resulted in complete loss of CIactivity reserves when glutathione levels are reduced,suggesting that the direct NO-mediated effects on CI aremore rate-limiting. Therefore complex I appears to havemajor control over respiration as its spare threshold capacityis lower than that of KGDH, i.e. the percentage by which itcan be inhibited before mitochondrial function is impaired ismuch less. Glutathione depletion appears to decrease thethresholds for both, suggesting that it is important formaintaining mitochondrial energy thresholds by protectingagainst different forms of oxidative stress to variousmitochondrial proteins integral to oxidative phosphorylation.This suggests that glutathione performs multiple antioxidantfunctions in terms of protecting the function of the dopam-inergic mitochondria which are lost during glutathionedepletion such as occurs in the PD SN. Mitochondrialenergy production as assessed by decreased ATP generationis indeed compromised in our glutathione-depleted cells. InPD, the ability of midbrain dopaminergic neurons to copewith energetic stress may also be compromised due to similarevents following early glutathione reduction rendering thenigrostriatal system particularly vulnerable to neurodegener-ation (Sofic et al. 1992).

What impact might glutathione depletion-mediated com-plex I inhibition via this NO+-mediated event have ondopaminergic cell viability? Generation of NO via thediethylamine/nitric oxide complex sodium (DEA/NO) haspreviously been reported to result in dopaminergic cell deathin fetal midbrain cultures which was only preventable bytreatment with thiol antioxidants such as GSH, GSH ethylester, or the GSH synthesis precursor N-acetyl-cysteine(NAC; Rodriguez-Martin et al. 2002). It was, however, notpreventable via treatment with peroxynitrite scavengers suchas uric or ascorbic acid or agents which prevent extra- orintracellular peroxynitrite formation, for example superoxidedismutase (SOD; EC 1.15.1.1) and 4,5-dihydroxy-1,3-ben-zene-disulfonic acid (Tiron). Although the exact cause of celldeath was not determined, these data imply that dopamin-ergic midbrain neurons are susceptible to NO toxicity via athiol oxidation-mediated mechanism which is independent of

Table 2 List of cysteine containing tetrapeptide sequences that have

been implicated in the formation of Fe-S clusters within complex I with

putative nitrosylation motifs given scores as in Table 1

Subunit Human Score Number Mouse Score Number

Ndufa8 AQCD PP 8(3) AQCD PP 8(3)

MLCR P MLCR P

RRCL RRCL

NKCA NGCA

RHCA SHCA

WTCI WTCL

RHCR PP RHCR PP

DECV DQCV

Ndufs1 QACE PP 12(4) QACE PP 12(4)

RFCY RFCY

GNCR PP GNCR PP

RMCL RMCL

AACA AACA

LDCP LDCP

PICD PP PICD PP

GECD PPP GECD PPP

TRCI TRCI

IQCT IQCT

TRCI TRCI

DICP DICP

Ndufs2 GDCY 3(1) GDCY 3(1)

YLCR P YLCR P

AQCL EQCL

Ndufs7 LACC LACC

ACCA ACCA

GSCA GSCA

PGCP PGCP

Ndufs8 ERCI 8(2) ERCI 8(2)

IACK P IACK P

KLCE PP KLCE PP

AICP AICP

TKCI TKCI

IYCG IYCG

GFCQ GFCQ

EACP EACP

Ndufv1 ESCG 4(1) ESCG 4(1)

GQCT GQCT

TPCR P TPCR P

TICA TICA

Ndufv2 QVCT 4(0) QVCT 4(0)

TPCM TPCM

VECL VECL

GACV GACV

1100 M. Hsu et al.


peroxynitrite formation. While we do not observe significantcell loss during brief glutathione depletion in our cell model,chronic glutathione depletion and continued loss of ATPwould likely eventually result in loss of cell viability. This iscurrently under investigation in the laboratory. Glutathionedepletion in the range of 50% prior to addition of normallyneurotrophic levels of NO has previously been demonstratedto result in increased dopaminergic cell death in fetalmidbrain cultures which is attenuated by co-incubation withthe antioxidant ascorbic acid (Canals et al. 2001).

In conclusion, our data suggest that direct nitrosation ofimportant cysteine residues within complex I by NO+ may bea major cause of its subsequent inhibition following totalglutathione depletion. This in turn is likely have a majorimpact on dopaminergic cell viability. This is particularlytimely information given that two publications from theDawson laboratory have recently demonstrated that nitrosa-tion of the Parkin protein, whose mutation has beenimplicated in early onset familial PD, can impact on itsactivity as an ubiquitin-proteosome E3 ligase (Chung et al.2004). This implies that protein S-nitrosation is likely animportant event impacting on dopaminergic cell deathassociated with the disease. Our study not only suggeststhat nitrosation as a consequence of early SN glutathionedepletion is the likely mechanism involved in the observedinhibition of complex I in PD which should in future aid inthe identification of potential nitrosation target sites withinthe complex, but also that therapeutic thiol treatments may beuseful in the disorder.

Acknowledgements

This work was supported in part by National Institutes of Health

grant R01 AG12141 (JKA) and a National Parkinson’s Foundation

post-doctoral grant (BS).

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Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's