Effects of hydrogen sulfide-releasing L-DOPA derivatives on glial activation: Potential for treating Parkinson disease

Received for publication, February 17, 2010, and in revised form, March 26, 2010 Published, JBC Papers in Press, April 5, 2010, DOI 10.1074/jbc.M110.115261

Moonhee Lee^‡, Valerio Tazzari^§, Daniela Giustarini^¶, Ranieri Rossi^¶, Anna Sparatore^§, Piero Del Soldato^ʈ,

Edith McGeer^‡, and Patrick L. McGeer^‡1

From the ^‡Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3,

Canada, the ^§Dipartimento di Scienze Farmaceutiche Pietro Pratesi, Universita` degli Studi di Milano, Via Mangiagalli 25, 20133,

Milano, Italy, the ^¶Department of Evolutionary Biology, Laboratory of Pharmacology and Toxicology, University of Siena,

via A. Moro 4, I-53100, Siena, Italy, and ^ʈCTG Pharma, Viale Gran Sasso 17, 20131 Milano, Italy

The main lesion in Parkinson disease (PD) is loss of substantia

nigra dopaminergic neurons. Levodopa (^L-DOPA) is the most

widely used therapy, but it does not arrest disease progression.

Some possible contributing factors to the continuing neuronal

loss are oxidative stress, including oxidation of ^L-DOPA, and

neurotoxins generated by locally activated microglia and astro-

cytes. A possible method of reducing these factors is to produce

L-DOPA hybrid compounds that have antioxidant and antiin-

flammatory properties. Here we demonstrate the properties of

four such ^L-DOPA hybrids based on coupling L-DOPA to four

different hydrogen sulfide-donating compounds. The donors

themselves were shown to be capable of conversion by isolated

substantia nigra zona compacta. Levodopa (L-DOPA) therapy is

the most widely used treatment because it helps to compensate

for the deficiency of dopamine. Whereas ^L-DOPA treatment is

very successful in the early stages of PD, it does not arrest dis-

ease progression, and in the long term there are unwanted side

effects, such as the development of dyskinesias (1–3). Many

previous studies have investigated the reasons for such long

term problems. One suggested mechanism is loss of neurons

induced by oxidative stress, including the oxidation of ^L-DOPA

(4–6). Another is persisting neuroinflammation caused by acti-

vated microglia and astrocytes (7–10). Hybrid molecules which

combine ^L-DOPA with antioxidant and antiinflammatory moi-

eties might therefore have therapeutic potential.

؊

mitochondria to H₂S or equivalent SH ions. This capability was

confirmed by in vivo results, showing a large increase in intra-

cerebral dopamine and glutathione after iv administration in

rats. When human microglia, astrocytes, and SH-SY5Y neuro-

blastoma cells were treated with these donating agents, they all

accumulated H₂S intracellularly as did their derivatives coupled

to ^L-DOPA. The donating agents and the L-DOPA hybrids

reduced the release of tumor necrosis factor-␣, interleukin-6,

and nitric oxide from stimulated microglia, astrocytes as well as

the THP-1 and U373 cell lines. They also demonstrated a neu-

roprotective effect by reducing the toxicity of supernatants from

these stimulated cells to SH-SY5Y cells. ^L-DOPA itself was with-

out effect in any of these assays. The H₂S-releasing L-DOPA

hybrid molecules also inhibited MAO B activity. They may be

useful for the treatment of PD because of their significant anti-

inflammatory, antioxidant, and neuroprotective properties.

Hydrogen sulfide (H₂S) has traditionally been regarded as

nothing more than a noxious gas with an extremely unpleasant

odor. But it is an essential body product. In solution it is a

powerful reducing agent with endogenous antioxidant and

neuroprotective properties. It is synthesized by the action of

two enzymes, cystathione ␤-synthase (CBS) and cystathione

␥-lyase (CGL). CBS is the main enzyme producing H₂S in the

brain (11, 12), whereas CGL is the main enzyme producing it in

vascular tissue (13). Much attention has been given to this mol-

ecule because it has multiple physiological and pathophysiolog-

ical functions in body organs. A protective role for H₂S in neu-

rons against oxidative stress has been reported (14). It has also

been demonstrated that H₂S induces alterations in calcium

channels [Ca^2ϩ]_iin astrocytes and microglia (15, 16) and

enhances both NMDA receptor-mediated neurotransmission

and long term potentiation (17).

Oxidative stress and neuronal cell death in PD is associated

with microglial activation, which involves the release of pro-

inflammatory cytokines and free radicals (9, 10). Recently, pro-

tective functions of H₂S against LPS-induced inflammation in

primary cultured and immortalized microglia have been

reported (12, 18, 19). Such a protective effect has been demon-

strated in vivo in the 6-OHDA rat model of Parkinson disease

(20).

The pathogenesis of Parkinson disease (PD)²results primar-

ily from loss of neurons, especially dopaminergic neurons of the

* This research was supported by the Pacific Alzheimer Research Foundation.

□S

The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. S1–S11.

¹To whom correspondence should be addressed: Kinsmen Laboratory of

Neurological Research, University of British Columbia, 2255 Wesbrook

Mall, Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-7377; Fax: 604-822-

7086; E-mail: mcgeerpl@interchange.ubc.ca.

To date there are no disease-modifying drugs for the treat-

ment of PD. As an approach to the development of next gener-

ation agents, we have prepared hybrid compounds designed to

²The abbreviations used are: PD, Parkinson disease; DMEM, Dulbecco’s

modified Eagle’s medium; PBS, phosphate-buffered saline; MTT, 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IL, interleukin;

TNF, tumor necrosis factor; MOPS, 4-morpholinepropanesulfonic acid;

OPT, o-phthalaldehyde; ANOVA, analysis of variance; CBS, cystathione

␤-synthase; LPS, lipopolysaccharide; IFN, interferon; LDH, lactate dehydro-

genase; MAO, monoamine oxidase.

17318 JOURNAL OF BIOLOGICAL CHEMISTRY

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H₂S-releasing L-DOPAs and Glial Activation

FIGURE 1. The structure and synthetic scheme of H₂S-releasing agents ACS 48, ACS 50, ACS 5, and ACS 81 and the H₂S-releasing L-DOPA derivatives,

ACS83, ACS84, ACS85, and ACS86.

combine the dopamine replacement properties of L-DOPA cell cultures: bacterial LPS (from Escherichia coli 055:B5) and

with the neuroprotective properties of H₂S donors.

human recombinant interferon-␥ (INF␥) (from Bachem Cali-

fornia, Torrance, CA). The following substances were used in

the assays: diaphorase (EC 1.8.1.4, from Clostridium kluyveri,

5.8 units/mg solid), p-iodonitrotetrazolium violet (INT), nico-

In this investigation, we synthesized four H₂S-releasing moi-

eties (ACS48, ACS50, ACS5, and ACS81) and examined

Ϫ

whether they release H₂S or equivalent SH ions in both glia

ϩ

and neurons. The sulfurated moieties were chosen among

those resembling the structures of known H₂S-releasing com-

pounds such as the dithiolethione ADT-OH (21) and diallyl-

disulfide (22). We coupled these moieties to ^L-DOPA methyl

ester through an amide linkage to create different lipophylic

compounds potentially able to release H₂S. The four hybrid

molecules were designated ACS83, ACS84, ACS85, and ACS86.

The structure of the four donors and the four hybrid molecules

are shown in Fig. 1. We made a pilot in vivo test with ACS84 to

confirm that these types of compounds can reach the brain.

We found that it reached the brain and that it produced an

increase of intracerebral dopamine and glutathione. We tested

the effects of all these compounds on prevention of neuronal

cell death induced by stimulation of four types of cultured

human glial cells: astrocytes, microglia, and the THP-1 and

U373 cell lines. NaSH was used as the standard H₂S donor for

comparative purposes. We found that these hybrid molecules

were able to release H₂S or equivalent ions from all cell types

tested, and from mitochondria isolated from U373 cells.

tinamide adenine dinucleotide (NAD ), and MTT (3-(4,5-di-

methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).

Chemistry—Some of the sulfurated intermediates were

already known and were prepared according to the literature.

All novel compounds synthesized were characterized by melt-

1

ing point (m.p.; Buchi apparatus), H NMR spectra (Varian

Mercuri 300VX spectrometer) and high-resolution mass spec-

tra (HRMS; APEX II ICR-FTMS Bruker Daltonics mass spec-

trometer, ESI). Log P were calculated by ChemDraw Ultra 9.0

software.

ACS48 (4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid) was

obtained by hydrolysis of the corresponding methyl ester,

which was synthesized as previously described by Adelaere

(23). ACS50 ([2-methoxy-4-(3-thioxo-3H-1,2-dithiol-5-yl)-

phenoxy]acetic acid, m.p. 198–200 °C) was obtained by acidic

hydrolysis of the corresponding methyl ester, which was

synthesized as described by Lozac’h and Mollier (24). ACS5

(1,3-dithiole-2-thioxo-4-carboxylic acid) was synthesized as

previously described by Dartigues et al. (25). ACS81 (3-(prop-

2-en-1-yldisulfanyl)propanoic acid) was synthesized as follows:

to a stirred solution of diallyl disulfide (2.4g; 13.6 mmol) in a

EXPERIMENTAL PROCEDURES

Materials—All reagents were purchased from Sigma unless

otherwise stated. The following substances were applied to the mixture of ether (10 ml) and methanol (20 ml), under nitrogen

JUNE 4, 2010•VOLUME 285•NUMBER 23

JOURNAL OF BIOLOGICAL CHEMISTRY 17319

H₂S-releasing L-DOPAs and Glial Activation

atmosphere at room temperature, was added a solution of (m, 2H). HMRS (ESI) m/z calculated for C₁₄H₁₃NO₅S₃Na

ϩ

3-mercaptopropanoic acid (0.49 g, 4.6 mmol) in ether (5 ml), [MϩNa] : 393.98481; found: 393.98524. Calc. log P: 1.68.

followed by a solution of 10 ^MNaOH (0.46 ml). The reaction

Methyl 2-(3-(allyldisulfanyl)propanamido)-3-(3,4-dihy-

mixture was stirred at room temperature for 24 h and, after droxyphenyl)propanoate, (ACS86)—Yield 50%. Semisolid; ¹H

evaporation of the solvents under reduced pressure, the crude NMR (DMSO-d₆): ␦ ϭ 8.74 (s, 1H, collapses with D₂O); 8.68 (s,

compound was taken up with ether and 1 ^NHCl. After separa- 1H, collapses with D₂O); 8.34 (d, J ϭ 7.92 Hz, 1H, collapses with

tion of the organic phase and evaporation of the ether, the res- D₂O); 6.59 (d, J ϭ 7.91 Hz, 1H); 6.54 (d, J ϭ 2.06 Hz, 1H); 6.41

idue was purified by column chromatography on silica gel, elut- (dd, J ϭ 2.06, 7.91 Hz, 1H,); 5.86–5.72 (m, 1H); 5.20–5.09 (m,

ing with CH₂Cl₂/CH₃COOC₂H₅(60:40). A colorless oil was 2H); 4.37–4.30 (m, 1H); 3.56 (s, 3H); 3.35 (d, J ϭ 7.33 Hz, 2H);

obtained (520 mg; yield 63%). ¹H NMR (CDCl₃): ␦ ϭ 5.91–5.77 2.82–2.64 (m, 4H); 2.49–2.46 (m, 2H). HMRS (ESI) m/z calcu-

ϩ

(m, 1H); 5.24–5.13 (m, 2H); 3.33 (d, J ϭ 7.30 Hz, 2H); 2.92 (t, J ϭ lated for C₁₆H₂₁NO₅S₂Na [MϩNa] : 394.07534; found:

6.90 Hz, 2H); 2.80 (t, J ϭ 6.90 Hz, 2H). HMRS (ESI) m/z cal- 394.07469. Calc. log P: 2.32.

ϩ

culated for C₆H₁₀O₂S₂Na [MϩNa] : 201.00144; found:

Animal Treatments and Sample Processing—Sprague-Daw-

201.00147.

ley rats (350–400 g) were purchased from Charles River. Rats

Synthesis of Sulfurated Derivatives of ^L-DOPA Methyl Ester received administration of ACS84 dissolved in 500 ␮l of

(ACS83, ACS84, ACS85, and ACS86)—^L-DOPA methyl ester PEG400 via the caudal vein. After 1 h, animals were anesthe-

hydrochloride (200 mg, 0.8 mmol), 1-hydroxybenzotriazole tized with pentobarbital (60 mg/kg), and blood was collected

(HOBt, 185 mg; 1.2 mmol) and 1-ethyl-3-(3-dimethylamino- from the abdominal aorta in tubes containing 50 mg/ml

propyl)carbodiimide hydrochloride (EDAC, 232 mg; 0.96 K₃EDTA and immediately processed. Plasma was obtained by

mmol) were added to a solution of the proper sulfurated com- centrifugation of blood at 10,000 ϫ g for 15 s and immediately

pounds (ACS48, or ACS50, or ACS5, or ACS81; 0.8 mmol) in deproteinized by addition of 4 volumes of acetonitrile (ACN).

anhydrous N,N-dimethylformamide (DMF) (4 ml) and, after The brain was then removed and cut longitudinally to obtain 2

the addition of triethylamine (0.22 ml; 1.6 mmol), the solution equal halves. One half was homogenized in 5 volumes of 4%

was stirred at room temperature for 24 h under a nitrogen (w/v) trichloroacetic acid containing 1 m^MK₃EDTA for the

atmosphere. After evaporation of DMF, the residue was dis- analyses of glutathione and dopamine. The other half was

solved in dichloromethane and the organic solution was homogenized in 5 volumes of a solution of 80% (v/v) acetoni-

washed sequentially with water, 1 ^NHCl, and water and finally trile to measure ACS84 and its metabolites. All animal manip-

dried with anhydrous Na₂SO₄and evaporated to dryness. The ulations were made in accordance with the European Commu-

crude compound was purified by flash chromatography (silica, nity guidelines for the use of laboratory animals. The

CH₂Cl₂/MeOH, 98:2).

experiments were authorized by the local ethical committee.

Methyl 3-(3,4-dihydroxyphenyl)-2-(4-(3-thioxo-3H-1,2-di-

HPLC Analyses of ACS84 Metabolites—For the determina-

thiol-4-yl)benzamido)propanoate (ACS83)—Yield 35%. M.p. tion of ACS84, ACS84-a, and ACS50, both brain and plasma

160–165 °C. ¹H NMR (DMSO-d₆): ␦ ϭ 9.21 (s, 1H); 8.81 (d, J ϭ samples were centrifuged to discard acetonitrile-denatured

7.63 Hz, 1H, collapses with D₂O); 8.74 (s, 1H, collapses with proteins (10,000 ϫ g for 2 min). The clear supernatants were

D₂O); 8.66 (s, 1H, collapses with D₂O); 7.85 (d, J ϭ 8.21 Hz, 2H); then diluted 1:1 with 0.05% trifluoroacetic acid, loaded onto an

7.65 (d, J ϭ 8.21 Hz, 2H); 6.65 (d, J ϭ 1.76 Hz, 1H); 6.59 (d, J ϭ HPLC (Zorbax Eclipse XDB-C18 column, 4.6 ϫ 150 mm, 5 ␮m,

8.21 Hz, 1H); 6.51 (dd, J ϭ 1.76, 8.21 Hz, 1H); 4.57–4.50 (m, Agilent Technologies, Milan, Italy) and separated by the appli-

1H); 3.62 (s, 3H); 2.99–2.85 (m, 2H). HMRS (ESI) m/z calcu- cation of a trifluoroacetic acid/acetonitrile solution: 0–9 min,

ϩ

lated for C₂₀H₁₇NO₅S₃Na [MϩNa] : 470.01611; found: 40% ACN in 0.05% (v/v) trifluoroacetic acid; 9–10 min,

470.01620. Calc. log P: 3.58.

40–90% ACN gradient. Analytes were detected at 435-nm

Methyl 3-(3,4-dihydroxyphenyl)-2-(2-(2-methoxy-4-(3- wavelength. The identity of the peak was determined by analyz-

thioxo-3H-1,2-dithiol-5-yl)phenoxy)acetoamido)propanoate, ing plasma samples spiked with the respective authentic com-

1

(ACS84)—Yield 53%. M.p. 153–155 °C. H NMR (DMSO-d₆): pounds. Calibration curves were generated in the 0.2–100 ␮M

␦ ϭ 8.79 (s, 1H, collapses with D₂O); 8.75 (s, 1H, collapses with range by addition of ACS50, ACS84, or ACS84-a to rat plasma

D₂O); 8.29 (d, J ϭ 7.91 Hz, 1H, collapses with D₂O); 7.85 (s, 1H); samples obtained from untreated animals.

7.42–7.37 (m, 2H); 6.77 (d, J ϭ 8.21 Hz, 1H); 6.61 (d, J ϭ 7.91 Hz,

HPLC Analyses of Dopamine and Glutathione for in Vivo

1H); 6.56 (d, J ϭ 2.05 Hz, 1H); 6.41 (dd, J ϭ 2.05, 7.91 Hz, 1H); Analysis—For the determination of GSH and dopamine, sam-

4.65–4.54 (m, 2H); 4.51–4.43 (m, 1H); 3.85 (s, 3H); 3.61 (s, 3H); ples were centrifuged to discard proteins (10,000 ϫ g for 2 min).

2.91–2.72 (m, 2H). HMRS (ESI) m/z calculated for Dopamine was measured on the clear supernatant by

ϩ

C₂₂H₂₁NO₇S₃Na [MϩNa] : 530.03724; found: 530.03660. UV-HPLC with fluorescence detection (excitation at 270 nm,

Calc. log P: 3.19.

emission at 320 nm, Zorbax Eclipse XDB-C18 column, 4.6 ϫ

Methyl 3-(3,4-dihydroxyphenyl)-2-(2-thioxo-1,3-dithiole-4- 150 mm, 5 ␮m). Separation was carried out by the application of

carboxamido)propanoate, (ACS85)—Yield 56%. M.p. 66–70 °C. an isocratic run: 5% (v/v) methanol in 0.05% (v/v) trifluoroace-

¹H NMR (DMSO-d₆): ␦ ϭ 9.24 (d, 1H, J ϭ 7.92 Hz, collapses tic acid (26). Another aliquot of the supernatant (0.1 ml) was

with D₂O); 8.76 (s, 1H, collapses with D₂O); 8.71 (s, 1H, col- brought to pH ϳ8.0 with 15 ␮l of 2 M Tris, and then 3 ␮l of 40

lapses with D₂O); 8.31 (s, 1H); 6.61–6.58 (m, 2H); 6.46 (dd, J ϭ m^Mmonobromobimane (mBrB) were added for the analysis of

1.76, 7.92 Hz, 1H); 4.47–4.39 (m, 1H); 3.61 (s, 3H); 2.96–2.76 GSH. After 10 min of incubation in the dark, samples were

17320 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 23•JUNE 4, 2010

H₂S-releasing L-DOPAs and Glial Activation

acidified and analyzed by HPLC (Zorbax Eclipse XDB-C18 col- excitation of 380 nm and an emission of 540 nm using a plate

umn, 4.6 ϫ 150 mm, 5 ␮m) as previously described (27).

Cell Culture and Experimental Protocols—The human

reader.

Preparation of Mitochondria from U373 Cells—Preparation

monocyte THP-1 and astrocytoma U373 cell lines were of functional mitochondria from human U373 cells was per-

obtained from the American Type Culture Collection (ATCC). formed as described previously (30). Briefly, U373 cells were

The human neuroblastoma SH-SY5Y cell line was a gift from detached from a tissue culture flask using a cell scraper and

Dr. R. Ross, Fordham University, NY. These cells were grown in were then transferred to 50 ml Falcon tubes for centrifugation

DMEM/F12 medium containing 10% fetal bovine serum (FBS), (2,000 rpm for 10 min) at 4 °C. After their supernatants were

100 international units/ml penicillin, and 100 ␮g/ml strepto- discarded, cells were resuspended in 30 ml of ice-cold mito-

mycin (Invitrogen, Carlsbad, CA) under humidified 5% CO₂chondria isolation buffer (10 m^MTris-MOPS, 1 mM EGTA, and

and 95% air. Human astroglial and microglial cells were isolated 0.2 ^Msucrose, pH 7.4), and homogenized with a glass/Teflon

from surgically resected temporal lobe tissue. The procedures potter homogenizer. The homogenates were transferred to 50

described previously for obtaining pure cultures of microglia ml Falcon tubes for centrifugation (2,000 rpm for 10 min) at

and astrocytes were followed (28).

4 °C. The supernatants were collected to perform high-speed

Experimental Protocol—Human astrocytes, U373 astrocy- centrifugation (30,000 rpm, 10 min) at 4 °C. After the superna-

toma cells and THP-1 cells (5 ϫ 10⁵cells), as well as human tants were discarded, the pellets were resuspended with 5 ml of

microglial cells (5 ϫ 10⁴cells) were seeded into 24-well plates in ice-cold mitochondria isolation buffer and centrifuged again

1 ml of DMEM/F12 medium containing 5% fetal bovine serum. (30,000 rpm, 10 min). The pellets including mitochondria were

The CBS inhibitor hydroxylamine (1 m^M) was added to inhibit resuspended very gently with 5 ml of PBS for H₂S measure-

endogenous production of H₂S by CBS. L-DOPA, (Ϫ)-deprenyl ments in the presence of H₂S releasers (ADT-OH, ACS48,

or H₂S-releasing moieties were then added at a concentration ACS50, ACS5, and ACS81).

of 10 ␮M. Incubation of the mixtures was carried out for 2, 4, 8,

Measurement of H₂S Levels—H₂S levels were measured using

or 12 h. The H₂S-releasing moieties included NaSH, ACS 48, a previously described method (21). To suppress endogenous

ACS50, ACS5, ACS81, and ADT-OH (21), as well as the hybrid production of H₂S by CBS, all experiments were done with 1

molecules ACS83, ACS84, ACS85, and ACS86. Cells were m^Mof the specific CBS inhibitor hydroxylamine added to the

washed with phosphate-buffered saline (PBS) twice and solutions. Two sets of experiments were conducted: one in

replated in 800 ␮l DMEM/F12 medium containing 5% FBS. The which the THP-1 cells and U373 cells were unstimulated and a

cells were then incubated at 37 °C for 2 days in the presence of second where they were stimulated with inflammatory media-

inflammatory stimulants. For microglia and THP-1 cells, the tors for 48 h. For THP-1 cells, the stimulation was LPS at 1

stimulants were LPS at 1 ␮g/ml and IFN␥ at 333 units/ml. For ␮g/ml and IFN␥ at 333 units/ml, and for U373 cells it was IFN␥

astrocytes and U373 cells, the stimulant was IFN␥ alone at 150 at 150 units/ml. The cells in each case were treated with 1 mM

units/ml. A companion set of cells was incubated in medium hydroxylamine plus NaSH, ADT-OH, ACS83, ACS84, ACS85,

without inflammatory stimulants. After incubation, the super- or ACS86 (10 ␮M each) for 2, 4, 8, and 12 h. Following treat-

natants (400 ␮l) were transferred to undifferentiated human ment, they were homogenized in 250 ␮l of ice-cold 100 mM

neuroblastoma SH-SY5Y cells (2 ϫ 10⁵cells per well). The cells potassium phosphate buffer (pH 7.4) containing trichloroacetic

were incubated for a further 72 h, and MTT and LDH assays acid (10% w/v). Zinc acetate (1% w/v, 250 ␮l) was injected to

performed as described below.

trap the generated H₂S. A solution of N,N-dimethyl-p-

SH-SY5Y Cell Viability Assays—The viability of SH-SY5Y phenylenediamine sulfate (20 ␮M; 133 ␮l) in 7.2 M HCl and

cells following incubation with glial cell supernatants was eval- FeCl₃(30 ␮M; 133 ␮l) in 1.2 M HCl was added. Absorbance at

uated by the LDH release and MTT assays as previously 670 nm of the resulting mixture (300 ␮l) was determined after

described in detail (29). For the LDH assay, the amount of LDH 10 min using a 96-well microplate reader (Bio-Rad). The H₂S

released was expressed as a percentage of the value obtained in concentration of each sample was calculated against a calibra-

comparative wells where cells were 100% lysed by 1% Triton tion curve of NaSH (1–250 ␮M) and results expressed as ␮mol/g

X-100. For the MTT assay, data are presented as a percentage of protein or nmol/ml.

the value obtained from cells incubated in fresh medium only.

GSH Level in Glial Cells in Vitro—The GSH level was

Measurement of TNF␣ and IL-6 Release—Cytokine levels assessed by the method of Hissin and Hilf (31). This assay

were measured in cell-free supernatants following 48 h of incu- detects reduced glutathione (GSH) by its reaction with

bation of THP-1 cells, U373 cells, microglial cells, or astrocytes. o-phthalaldehyde (OPT) at pH 8.0. Cells (10⁶) in 1.5-ml tubes

The cell stimulation protocols were the same as used for mea- were washed twice with PBS, and treated with 200 ␮l of 6.5%

suring H₂S generation. Quantitation was performed with (w/v) trichloroacetic acid. The mixture was incubated on ice for

ELISA detection kits (Peprotech, NJ) following protocols 10 min and centrifuged (13,000 rpm, 1 min). The supernatant

described by the manufacturer.

was discarded, and the pellets were resuspended in 200 ␮l of

Ϫ

Measurements of Nitrite Release—Accumulation of NO₂as ice-cold 6.5% (w/v) trichloroacetic acid and centrifuged again

an indicator of NO synthesis was assayed by the standard Griess (13,000 rpm, 2 min). Supernatants (7.5 ␮l) were transferred to

reaction. After stimulation of cells for 48 h, the media were 96-well plates containing 277.5 ␮l phosphate-EDTA buffer (pH

centrifuged, and the cell-free supernatants mixed with an equal 8.0) in 1 ^MNaOH solution. Then 15 ␮l of OPT (1 mg/ml in

volume of Griess reagent (Sigma). Samples were incubated at methanol) was added. The reaction mixture was incubated in

room temperature for 15 min, and the fluorescence read at an the dark at room temperature for 25 min. The fluorescence at

JUNE 4, 2010•VOLUME 285•NUMBER 23

JOURNAL OF BIOLOGICAL CHEMISTRY 17321

H₂S-releasing L-DOPAs and Glial Activation

TABLE 1

Levels of intact molecule and its metabolites in plasma and brain 1 h after iv rat administration of 40 mg/kg ACS 84

Plasma

ACS84-a

Brain

ACS84-a

ACS84

ACS50

ACS84

ACS50

␮M mean Ϯ S.E.

6.7267 Ϯ 0.8933

␮M mean Ϯ S.E.

0.3497 Ϯ 0.0568

1.1137 Ϯ 0.0867^a

58.9333 Ϯ 3.6988

0.1230 Ϯ 0.0227

1.3227 Ϯ 0.2476

^aValues are mean Ϯ S.E., n ϭ 4.

TABLE 2

Dopamine, L-DOPA, and GSH levels in brain and/or plasma 1 h after iv rat administration of 40 mg/kg ACS84 or an equimolar dose of L-DOPA

Values are mean Ϯ S.E., n ϭ 4. One-way ANOVA test was carried out to evaluate the significance of differences.

Plasma

Brain

L-DOPA

␮M

Treatments

Dopamine

L-DOPA

␮M

Dopamine

GSH

␮M

ACS84

2.17 Ϯ 0.52^a

29.2 Ϯ 3.8^b

0

13.2 Ϯ 1.3^a

9.60 Ϯ 0.08^b

5.97 Ϯ 0.6

0.277 Ϯ 0.2775^a

2.00 Ϯ 0.775^b

0

2402 Ϯ 39.3^a

1479 Ϯ 34

1670 Ϯ 60

L-DOPA

Vehicle

10.2 Ϯ 2.8^b

0

^ap Ͻ 0.01 for ACS84 group compared with vehicle group.

^bp Ͻ 0.01 for L-DOPA group compared with ACS84 group.

350 nm excitation/420 nm emission was measured in a multi- and ACS81) under extracellular conditions. We tested

well plate reader. The concentration was calculated from a

standard curve using a serial dilution of reduced GSH.

ActivityofMonoamineOxidaseAandB—Activitiesofmono-

amine oxidase A and B (MAO A and MAO B, respectively) after

SH-SY5Y cells were exposed to glial-conditioned medium for 1

day were measured using a kit (Amplex Red monoamine oxi-

dase kit, Molecular Probes Inc., Eugene, OR). Experiments were

performed as described by the manufacturer.

release from PBS, the DMEM/F12 culture medium, and

human serum. We then compared this with the rate of

uptake and cleavage intracellularly in THP-1, U373, and

SH-SY-5Y cells (Fig. 2).

In PBS and the DMEM/F12 medium, H₂S was released very

slowly, reaching only 10% of the total amount available after

48 h (see Fig. 2B for ACS50 as a representative in DMEM/F12

medium and for ACS50 in PBS). Human serum caused a more

rapid release with 50% of the potential being attained in 4.5–18

h. (ADT-OH, 4.5 h; ACS5,6 h; ACS 48, 6 h; ACS 50, 12 h; and

ACS 81, 18 h) indicating the presence of weakly active cleaving

enzymes in human serum. In contrast, NaSH, the model H₂S

donor, released its full potential immediately. It then showed a

slow decay, declining by about 10% over 48 h (see supple-

Data Analysis—The significance of differences between data

sets was analyzed by Student’s t test and one-way or two-way

ANOVA. Multiple group comparisons were followed by a post-

hoc Bonferroni test.

RESULTS

First, we evaluated the release rate in vivo of the H₂S donor

from ACS84, a typical ^L-DOPA hybrid compound, adminis-

tered intravenously (40 mg/kg) into rats. Table 1 demonstrates

the formation of the main metabolites of ACS84. After 1 h,

almost all of the ACS84 had disappeared from plasma with the

concomitant appearance of both its demethylated derivative

(ACS84-a) and of its dithiolethione moiety (ACS50). Measure-

ments performed at earlier times from the treatments indicate

that demethylation of ACS84 occurs rapidly, whereas the cleav-

age of the amide bond between dithiolethione and ^L-DOPA

occurs more slowly (data not shown). Low micromolar or sub-

micromolar concentrations of ACS84 and its main metabolites

were found in brain 1 h after administration.

Ϫ

mental Fig. S1, B , D , and F ), indicating minor decay of SH ions.

These data demonstrate that the H₂S donating molecules are

stable extracellularly, and, if administered in vivo, should be

able to reach their target cells relatively intact.

Fig. 2 also indicates what happens when the H₂S donors do

reach their target cells. The data show intracellular levels of H₂S

in THP-1, SH-SY5Y, and U373 cells after being exposed to 10

␮M of the H₂S donors. H₂S generated from the donor molecules

slowly increased in the cytoplasm of THP-1 and SH-SY5Y cells

over 48 h. Fig. 2 shows data from ACS50 as a representative of

the S-donor compounds. All the H₂S-donor compounds gave

highly similar results. For U373 cells, the conversion was more

robust than for THP-1 and SY-SY5Y cells with a maximum

being reached by 12 h, after which there was a slow decline (Fig.

2A). The intracellular H₂S levels reflect the net effect of at least

three mechanisms: uptake of donors into the cells; intracellular

cleavage of the molecules; and intracellular metabolism of the

H₂S generated. The other H₂S-releasing compounds ACS48,

ACS5, and ACS81 showed the same release kinetics in all cells

tested (see supplemental Fig. S1 ), as did the S-DOPA derivative

ACS83 (see supplemental Fig. S2).

Dopamine levels in brain were increased 2.2-fold by treat-

ment with ACS84. Interestingly, GSH, a known antioxidant and

neuroprotective agent (32) was increased 1.4-fold by this treat-

ment. Although treatment with an equimolar dose of ^L-DOPA

also increased dopamine levels in brain, the increase was only

by 1.6-fold, and there was no increase in GSH (Table 2). These

data encouraged us to pursue in vitro experiments to determine

whether H₂S-releasing L-DOPA hybrid compounds can lead to

accumulation of H₂S in both glia and SH-SY5Y cells, and what

effects this release might produce.

These data establish that all the moieties are metabolized

within each cell type to generate H₂S. To explore a possible

We commenced by examining the rate of cleavage of the

H₂S-releasing moieties (ADT-OH, ACS48, ACS50, ACS5, mechanism, we purified functional mitochondria from U373

17322 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 23•JUNE 4, 2010

H₂S-releasing L-DOPAs and Glial Activation

FIGURE 2. Intracellular (A) and extracellular (B) H₂S levels (nmol/ml) after treatment with 10 ␮M ACS50 in the presence of 1 mM hydroxylamine. Values

are mean Ϯ S.E., n ϭ 4.

for 2 days) or U373 cells (IFN␥ for 2 days). This treatment

caused a huge decrease in [GSH]_i(ϳ90% decrease, p Ͻ 0.01).

NaSH and the four H₂S-releasing S-DOPAs significantly atten-

uated this decrease (p Ͻ 0.01). Nevertheless, the values were

still significantly lower than those obtained from control media.

These data establish that H₂S generated from the donor com-

pounds is significantly converted into the antioxidant [GSH]_i

and that this [GSH]_iis significantly depleted by exposure to

supernatants from glial cells that have received inflammatory

stimulation.

Fig. 4, B and C demonstrates the changes in MAO A and B in

SH-SY5Y cells in the presence of NaSH, ^L-DOPA, and the four

H₂S-releasing S-DOPAs. SH-SY5Y cells express both MAO A

and MAO B, the latter accounting for 75% of the total MAO

activity. Treatment with NaSH or four different S-DOPAs (10

FIGURE 3. H₂S levels (␮mol/g protein) generated by isolated functional

mitochondria in human U373 cells after treatment with 10 ␮M ADT-OH,

ACS48, ACS50, ACS5, ACS81, or (؊)-deprenyl. Values are mean Ϯ S.E.,

n ϭ 4.

␮M each) for 8 h reduced the activity of MAO B, but not MAO

A. There was no effect of ^L-DOPA. The decreases were about

85% (p Ͻ 0.01) after exposure of SH-SY5Y cells to media from

unstimulated THP-1 or U373 cells and about 65% after expo-

sure to medium from THP-1 or U373 cells stimulated for 24 h

(p Ͻ 0.01). These data establish that all the S-DOPAs are selec-

tive MAO B inhibitors.

cells. All mitochondria contain molecules which have a high

reducing potential such as NADH, FADH₂, and cytochromes.

As shown in Fig. 3, all the donor moieties, but not (Ϫ)-depre-

nyl, a classical inhibitor of monoamine oxidase B, were metab-

olized to release H₂S within 60 min by the mitochondria. Very

similar results were obtained with the four different S-DOPA

compounds (see supplemental Fig. S3). The data indicate a

potential mechanism by which H₂S is generated intracellularly

from the donor moieties.

We next investigated the effects of 1, 3, 10, 30, and 50 ␮M

concentrations of the S-DOPA derivatives, as well as the H₂S

donors ADT-OH and NaSH on glial-mediated neurotoxicity.

Fig. 5 shows the effect on SH-SY5Y viability of adding NaSH,

ADT-OH, ^L-DOPA, (Ϫ)-deprenyl, ACS83, ACS84, ACS85, or

ACS86 to LPS/IFN␥-activated THP-1 cells (5A) or IFN␥-acti-

vated U373 cells (5B) at a standard concentration of 10 ␮M. It

was found that the H₂S-releasing agents ADT-OH and NaSH,

but not ^L-DOPA or (Ϫ)-deprenyl, attenuated the neurotoxicity

in an incubation time-dependent manner. This is shown both

by the MTT assay (upper panels) and LDH assay (lower panels).

The four S-DOPA derivatives ACS83, ACS84, ACS85, and

ACS86 were comparably protective to NaSH and ADT-OH.

Complete data showing the results at 1, 3, 10, 30, and 50 ␮M , and

2, 4, 8, and 12 h pretreatment are shown in supplemen-

Within cells, the most important reducing agent is gluta-

thione (GSH). To determine whether the H₂S donors were

affecting GSH levels, we measured [GSH]_iin SH-SY5Y cells

exposed to the four different S-DOPAs (10 ␮M each) for 8 h.

Equal concentrations of ^L-DOPA and NaSH were used as

negative and positive controls, respectively. The results are

shown in Fig. 4A. Treatment with NaSH and the S-DOPAs

increased [GSH]_iin SH-SY5Y cells under normal conditions by

ϳ1.5-fold (p Ͻ 0.01). We then exposed the cells for 1 day to

conditioned medium from stimulated THP-1 cells (LPS/IFN␥ tal Figs. S4 and S5. Again, the results were highly similar for all

JUNE 4, 2010•VOLUME 285•NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 17323

H₂S-releasing L-DOPAs and Glial Activation

FIGURE 4. A, effect of direct treatment with NaSH, L-DOPA, ACS83, ACS84, ACS85, or ACS86 on GSH levels in SH-SY5Y cells. After SH-SY5Y cells were treated with

NaSH, L-DOPA, ACS83, ACS84, ACS85, or ACS86 (10 ␮M each) for 8 h and subsequently exposed to THP-1 or U373 conditioned medium (CM) for 1 day, they were

extracted to measure GSH levels. B and C, effect of direct treatment with NaSH, ADT-OH (ADT), L-DOPA, ACS83, ACS84, ACS85, or ACS86 on activities of MAO A

and MAO B in SH-SY5Y cells exposed to stimulated THP-1 or U373-conditioned medium (CM). After SH-SY5Y cells were treated with NaSH, L-DOPA, ACS83,

ACS84, ACS85, or ACS86 (10 ␮M each) for 8 h and subsequently exposed to THP-1 or U373 CM for 1 day, they were extracted to measure activities of MAO A (B)

and MAO B (C). Notice that none of the compounds inhibited MAO A, but all of them significantly inhibited MAO B except for L-DOPA. Notice also that there was

significantly less inhibition when the SH-SY5Y cells were exposed to CM. Values are mean Ϯ S.E., n ϭ 4. Two-way ANOVA was initially carried out to test the

significance among the treatment groups. The significance of differences was then adjusted by applying the Bonferroni test for multiple comparisons. *, p Ͻ

0.01 for NaSH or all the ACS-treated groups compared with untreated group; **, p Ͻ 0.01 exposed to media from stimulated THP-1 or U373 compared with

control media; and ***, p Ͻ 0.01 exposed to stimulated THP-1 or U373 media treated with NaSH or all the ACS compounds compared with stimulated media

only.

the SH-donors and all the S-DOPA derivatives in their concen-

tration and incubation time dependence.

Inflammatory stimulation of microglia or THP-1 cells causes

them to release the inflammatory cytokines TNF␣ and IL-6, as

The 8-h time period shown in Fig. 5 demonstrated a robust well as to generate neurotoxic nitrite ions. Fig. 7 shows the

effect of THP-1 and U373 cells, so it was deemed optimum effect of treatment with NaSH, ADT-OH, and the four S-DOPA

for showing comparative effects using human microglia compounds (10 ␮M each, 8 h of preincubation) on THP-1

and astrocytes. It was found that the protective effect of pre- release of TNF␣ (Fig. 7A), IL-6 (Fig. 7C), and nitrite ions (Fig.

treatment with NaSH and the S-DOPAs (10 ␮M, 8 h) was 7E), and on human microglial release of TNF␣ (Fig. 7B), IL-6

comparable in human microglia and astrocytes to that found (Fig. 7D) and nitrite ions (Fig. 7F). There was a substantially

for THP-1 and U373 cells (Fig. 6A: microglia and 6B: astro- lower release of nitrite ions by stimulated microglia compared

cytes). MTT data are shown in the upper panels and LDH with THP-1 cells, even allowing for the 10-fold lower number of

release data in the lower panels. More detailed experimental microglial cells that were seeded. This is perhaps related to the

data (3, 10, and 50 ␮M; 8 h of preincubation) showing com- reported poor ability of human microglia to express iNOS. Nev-

parable effects at differing concentrations are shown in ertheless there were significant differences between the release

supplemental Fig. S6.

of these materials in both types of cells: (1) between stimulated

17324 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 23•JUNE 4, 2010

H₂S-releasing L-DOPAs and Glial Activation

and unstimulated cells and (2)

between stimulated cells that were

untreated and stimulated cells that

were treated with NaSH, ADT-OH,

or any one of the S-DOPA com-

pounds (p

Ͻ

0.01). However,

L-DOPA or (Ϫ)-deprenyl did not

affect the release of any of these

proinflammatory mediators (Fig. 7).

Fig. 8 shows comparable data for

IL-6 release from U373 cells (Fig.

8A) and cultured astrocytes (Fig.

8B). Cells were activated with IFN␥

as described under “Experimental

Procedures” and were then treated

similarly to the THP-1 and micro-

glial cells as shown in Fig. 5. In both

types of cells significant differences

were found: (1) for the release of

IL-6 from stimulated compared

with unstimulated cells and (2) for

stimulated cells treated with NaSH,

ADT-OH, or the four S-DOPA

compounds compared with stimu-

lated cells that were untreated (p Ͻ

0.01). Complete data showing a sim-

ilar concentration dependence of

the four donor moieties and the four

S-DOPA derivatives on H₂S release

(3, 10, and 50 ␮M ; 8 h pretreatment)

are shown in supplemental Figs.

S7 and S8. Supplemental Fig. S9

shows that the viability of THP-1,

U373, microglia, and astrocytes was

unaffected by any of the eight S-do-

nating agents. Supplemental Fig.

S10 shows that the neuroprotective

effects were additive when there was

a combination of two S-DOPA

derivatives (i.e. ACS 83 ϩ 84, ACS

84 ϩ 85, ACS 84 ϩ 86) compared

with the same derivatives alone.

FIGURE5. EffectontheviabilityofSH-SY5Ycellsfollowingtreatmentwithsupernatantsfromstimulated

THP-1 cells (A) or U373 cells (B) that had been exposed to 10 ␮M of the neuroprotective moieties as

described under “Experimental Procedures.” These were NaSH, the H₂S-releasing dithiol-thione moiety

ADT-OH, ACS48, ACS50, ACS5, ACS81, ACS83, ACS84, ACS85, or ACS86 pre-administered for (ꢀ, 0 h) (o, 2 h), (z,

4 h), (p, 8 h), and (f, 12 h). L-DOPA or (Ϫ)-deprenyl, which were without effect, served as the negative control

and NaSH as the positive control. Notice that the SH donors were equally protective and were comparable to

the the standard donor NaSH. Upper panels show MTT results whereas lower panels show LDH results. Values

are mean Ϯ S.E., n ϭ 4. Two-way ANOVA was carried out to test the significance of differences. Multiple

comparisons were followed with post-hoc Bonferroni tests. The time-dependent values were significantly

different from control (p Ͻ 0.01) for NaSH and each of the S-donating compounds.

DISCUSSION

In the present study, we exam-

ined the antioxidant and antiinflam-

matory properties of four H₂S

donors (ACS48, ACS50, ACS5, and

ACS81 as well as ADT-OH), and

four ^L-DOPA hybrid compounds

synthesized from the four donors

(ACS83, ACS84, ACS85, and

ACDS86). They all demonstrated

therapeutic potential by being taken

up by human microglia, and astro-

cytes, as well as the humanTHP-1

U373 cell lines and then generating

intracellular H₂S. The H₂S they gen-

FIGURE 6. Effect of treatment with NaSH, ADT-OH, L-DOPA, (؊)-deprenyl, ACS48, ACS50, ACS5, ACS81,

ACS83, ACS84, ACS85, or ACS86 (10 ␮M each, 8 h of preincubation) on SH-SY5Y cell viability changes

induced by activated human microglia (A) or activated astrocytes (B) as followed by MTT (upper panels)

andLDHrelease (lower panels) assays. Valuesaremean Ϯ S.E., n ϭ 4. One-wayANOVA was carried out totest

the significance of differences. Multiple comparisons were followed with post-hoc Bonferroni tests where

appropriate. *, p Ͻ 0.01 comparing the stimulated (ST) with the non-stimulated (NO-ST) group and **, p Ͻ 0.01

comparing stimulated (ST) group with the S-DOPA treatment groups.

JUNE 4, 2010•VOLUME 285•NUMBER 23

JOURNAL OF BIOLOGICAL CHEMISTRY 17325

H₂S-releasing L-DOPAs and Glial Activation

sequent neurotoxic effects (32). The

compounds described here could

help counteract the consequences

of depressed GSH production. Our

pilot in vivo data with ACS 84

showed that it reached the brain and

was substantially metabolized as

early as 1 h after iv administration to

a rat. There was a more than a 2-fold

increase in brain dopamine and a

1.4-fold increase in GSH. This dem-

onstrates that a significant amount

of the intact, and very lipophilic

ACS84, crosses the blood brain bar-

rier and is hydrolyzed in the brain

into its two components ^L-DOPA

and ACS50. Moreover the concom-

itant inhibition of the dopamine-

metabolizing enzyme MAO B, as

had previously been shown for

dithiolethiones (33), could contrib-

ute to sustain the high concentra-

tion of dopamine in brain. The

increase of GSH presumably results

from the H₂S generated by metabo-

lism of the donor moiety. While

very preliminary, these data demon-

strate that these ^L-DOPA hybrids

may have therapeutic potential by

enhancing dopamine and GSH

levels.

Earlier studies have indicated that

H₂S is a reducing agent that can

increase levels of intracellular gluta-

thione (14) thereby inhibiting oxi-

dative stress. It also decreases levels

of peroxynitrite-derived nitrated

proteins (34). Thus H₂S might block

oxidative stress-mediated cell death

in PD by directly or indirectly

detoxifying free radicals generated

FIGURE 7. Effect of treatment with NaSH, ADT-OH, L-DOPA, (؊)-deprenyl, ACS48, ACS50, ACS5, ACS81,

ACS83, ACS84, ACS85, or ACS86 (10 ␮M each, 8 h of preincubation) on the released levels of TNF␣ (A, B), from oxidized ^L-DOPA compounds.

IL-6 (C, D) and nitrite ions (E, F) from THP-1 cells (A, C, E) or human microglia (B, D, F). Values are mean Ϯ

As far as neuroinflammation is

S.E., n ϭ 4. One-way ANOVA was carried out to test the significance of differences. Multiple comparisons were

followed with post-hoc Bonferroni tests where appropriate. *, p Ͻ 0.01 comparing the stimulated group with

the non-stimulated group (NO-ST) and **, p Ͻ 0.01 comparing the stimulated group (ST) with the S-DOPA

treatment groups.

concerned, it is well known that

areas affected in PD, especially the

substantia nigra, are characterized

by the presence of activated micro-

erated acted in part to enhance levels of the classical antioxi- glia and activated astrocytes (4, 7–10, 35, 36). There is compa-

dant GSH. They also inhibited MAO B. Moreover, they acted as rable glial activation in animal models of PD (37). Evidence that

antiinflammatory compounds by ameliorating the neurotoxic these reactive glial cells contribute to the neuronal degenera-

effects of glial cell supernatants toward SH-SY5Y cells. Addi- tion comes from epidemiological studies where persons taking

tionally, they inhibited release of the proinflammatory media- NSAIDs are reported to be relatively spared from PD (38, 39)

tors TNF␣, IL-6, and nitrite ions (Figs. 7 and 8). The com- especially if combined with coffee (40). Such protection is also

pounds were equipotent, but ^L-DOPA was without effect in all reported for animal models of PD (37, 41).

of these assays.

The MPTP phenomena may be particularly revealing with

We have previously reported that depleting intracellular respect to the potential consequences of inducing SN inflam-

GSH by inhibiting its synthetic enzyme ␥-glutamylcysteine syn- mation. Drug addicts who were originally exposed to MPTP

thase induces an inflammatory reaction in glial cells with con- developed a relentlessly progressive parkinsonian syndrome.

17326 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 23•JUNE 4, 2010

H₂S-releasing L-DOPAs and Glial Activation

exposed to the inflammatory effects

of THP-1- or U373 cell-conditioned

medium. This could be due to con-

sumption of the generated H₂S

in stimulated cells, presumably

through oxidative reactions.

In conclusion, our data demon-

strate that H₂S not only has anti-

inflammatory activity against the

glial toxicity which may be associ-

ated with the pathogenesis of PD

(36), but may also reduce the stress

induced by ^L-DOPA oxidation (14).

Furthermore, by inhibiting MAO B,

FIGURE 8. Effect of treatment with NaSH, ADT-OH, L-DOPA, (؊)-Deprenyl, ACS48, ACS50, ACS5, ACS81,

ACS83, ACS84, ACS85, or ACS86 (10 ␮M each, 8 h of preincubation, protocol 1) on release of IL-6 from

U373 cells(A)orhumanastrocytes (B). Valuesaremean Ϯ S.E., n ϭ 4. One-wayANOVA was carried out totest

the significance of differences. Multiple comparisons were followed with post-hoc Bonferroni tests where

appropriate. *, p Ͻ 0.01 comparing the unstimulated (NO-ST) group with the stimulated group. **, p Ͻ 0.01

comparing the stimulated (ST) group with the S-DOPA-treated groups.

H₂S-releasing L-DOPA compounds

could help restore the disease-

depleted dopamine levels. The

H₂S-releasing L-DOPA derivatives

(S-DOPAs) described here repre-

Postmortem studies showed persistent inflammation up to sent compounds that can reach the brain and, in cells under

Ϫ

17 years following their last exposure to the toxin (35). This stress, deliver ameliorating SH ions in a time-dependent man-

human experience was duplicated in monkeys where inflam- ner. They have properties that make them candidates for future

mation of the SN was demonstrated up to 14 years after their treatment of PD. However the long term consequences of their

last MPTP exposure (8). This suggests that inflammation of use will require much further study.

the SN, once initiated, may be self sustaining. If this is the case,

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17328 JOURNAL OF BIOLOGICAL CHEMISTRY

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Effects of hydrogen sulfide-releasing L-DOPA derivatives on glial activation: Potential for treating Parkinson disease

DOI: 10.1074/jbc.M110.115261

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