Levodopa deactivates enzymes that regulate thiol-disulfide homeostasis and promotes neuronal cell death: implications for therapy of Parkinson's disease

Abstract

Parkinson's disease (PD), characterized by dopaminergic neuronal loss, is attributed to oxidative stress, diminished glutathione (GSH) levels, mitochondrial dysfunction, and protein aggregation. Treatment of PD involves chronic administration of Levodopa (l-DOPA) which is a pro-oxidant and may disrupt sulfhydryl homeostasis. The goal of these studies is to elucidate the effects of l-DOPA on thiol homeostasis in a model akin to PD, i.e., immortalized dopaminergic neurons (SHSY5Y cells) with diminished GSH content. These neurons exhibit hypersensitivity to l-DOPA-induced cell death, which is attributable to concomitant inhibition of the intracellular thiol disulfide oxidoreductase enzymes. Glutaredoxin (Grx) was deactivated in a dose-dependent fashion, but its content was unaffected. Glutathione disulfide (GSSG) reductase (GR) activity was not altered. Selective knockdown of Grx resulted in an increased level of apoptosis, documenting the role of the Grx system in neuronal survival. l-DOPA treatments also led to decreased activities of thioredoxin (Trx) and thioredoxin reductase (TR), concomitant with diminution of their cellular contents. Selective chemical inhibition of TR activity led to an increased level of apoptosis, documenting the Trx system's contribution to neuronal viability. To investigate the mechanism of inhibition at the molecular level, we treated the each isolated enzyme with oxidized l-DOPA. GR, Trx, and TR activities were little affected. However, Grx was inactivated in a time- and concentration-dependent fashion indicative of irreversible adduction of dopaquinone to its nucleophilic active-site Cys-22, consistent with the intracellular loss of Grx activity but not Grx protein content after l-DOPA treatment. Overall l-DOPA is shown to impair the collaborative contributions of the Grx and Trx systems to neuron survival.

Figure 1. Diminution of [GSH] in SHSY5Y cells leads to increased sensitivity to L-DOPA and increased cell death

(A) Bar graph displaying relative cell viability as measured by the MTT assay (see Methods) when treated with L-DOPA for 24 hr. Solid bars represent cells with replete GSH. Open bars indicate cells treated with 0.1mM BSO. BSO treatment showed a 3-fold increase in sensitivity to L-DOPA. * represent significant difference compared to control, p<0.01 (n = 3). (B) Dose-Response Curves for L-DOPA dependent cell death (trypan blue). SHSY5Y cells were treated with L-DOPA for 24 hr and trypan blue was used to measure cell death, as described in Methods. Treatment in the presence of BSO led to ~3-fold sensitization of SHSY5Y cells to L-DOPA induced cell death. ■ Symbols represent BSO + L-DOPA treated cells; ◆ represent L-DOPA only treated samples. (C) Dose-Response Curves for L-DOPA dependent apoptosis (Hoechst staining). SHSY5Y cells were treated with L-DOPA for 24 hr and apoptosis was measured using Hoechst 33342, determining the extent of chromatin condensation (see Methods). Samples were counted in triplicate in a blinded fashion. ■ Symbols represent BSO + L-DOPA treated cells; ◆ represent L-DOPA only treated samples. The curves in Figure 1 were fit to the % apoptosis vs. [L-DOPA] data according to the following equation, minimizing the least squares deviation: $$\%\text{apoptosis}={\text{Effect}}_{\max }\ast {[\mathrm{L}-\mathrm{DOPA}]}^{\mathrm{n}}∕{\left({\mathrm{EC}}_{50}\right)}^n+{[\mathrm{L}-\mathrm{DOPA}]}^{\mathrm{n}},\text{where}\mathrm{n}\text{is equivalent to the Hill coefficient}$$ The EC₅₀ values for L-DOPA induced apoptosis calculated from the fitted curves are 49 μM (plus BSO) and 127 μM (minus BSO).

Figure 1. Diminution of [GSH] in SHSY5Y cells leads to increased sensitivity to L-DOPA and increased cell death

(A) Bar graph displaying relative cell viability as measured by the MTT assay (see Methods) when treated with L-DOPA for 24 hr. Solid bars represent cells with replete GSH. Open bars indicate cells treated with 0.1mM BSO. BSO treatment showed a 3-fold increase in sensitivity to L-DOPA. * represent significant difference compared to control, p<0.01 (n = 3). (B) Dose-Response Curves for L-DOPA dependent cell death (trypan blue). SHSY5Y cells were treated with L-DOPA for 24 hr and trypan blue was used to measure cell death, as described in Methods. Treatment in the presence of BSO led to ~3-fold sensitization of SHSY5Y cells to L-DOPA induced cell death. ■ Symbols represent BSO + L-DOPA treated cells; ◆ represent L-DOPA only treated samples. (C) Dose-Response Curves for L-DOPA dependent apoptosis (Hoechst staining). SHSY5Y cells were treated with L-DOPA for 24 hr and apoptosis was measured using Hoechst 33342, determining the extent of chromatin condensation (see Methods). Samples were counted in triplicate in a blinded fashion. ■ Symbols represent BSO + L-DOPA treated cells; ◆ represent L-DOPA only treated samples. The curves in Figure 1 were fit to the % apoptosis vs. [L-DOPA] data according to the following equation, minimizing the least squares deviation: $$\%\text{apoptosis}={\text{Effect}}_{\max }\ast {[\mathrm{L}-\mathrm{DOPA}]}^{\mathrm{n}}∕{\left({\mathrm{EC}}_{50}\right)}^n+{[\mathrm{L}-\mathrm{DOPA}]}^{\mathrm{n}},\text{where}\mathrm{n}\text{is equivalent to the Hill coefficient}$$ The EC₅₀ values for L-DOPA induced apoptosis calculated from the fitted curves are 49 μM (plus BSO) and 127 μM (minus BSO).

Figure 1. Diminution of [GSH] in SHSY5Y cells leads to increased sensitivity to L-DOPA and increased cell death

(A) Bar graph displaying relative cell viability as measured by the MTT assay (see Methods) when treated with L-DOPA for 24 hr. Solid bars represent cells with replete GSH. Open bars indicate cells treated with 0.1mM BSO. BSO treatment showed a 3-fold increase in sensitivity to L-DOPA. * represent significant difference compared to control, p<0.01 (n = 3). (B) Dose-Response Curves for L-DOPA dependent cell death (trypan blue). SHSY5Y cells were treated with L-DOPA for 24 hr and trypan blue was used to measure cell death, as described in Methods. Treatment in the presence of BSO led to ~3-fold sensitization of SHSY5Y cells to L-DOPA induced cell death. ■ Symbols represent BSO + L-DOPA treated cells; ◆ represent L-DOPA only treated samples. (C) Dose-Response Curves for L-DOPA dependent apoptosis (Hoechst staining). SHSY5Y cells were treated with L-DOPA for 24 hr and apoptosis was measured using Hoechst 33342, determining the extent of chromatin condensation (see Methods). Samples were counted in triplicate in a blinded fashion. ■ Symbols represent BSO + L-DOPA treated cells; ◆ represent L-DOPA only treated samples. The curves in Figure 1 were fit to the % apoptosis vs. [L-DOPA] data according to the following equation, minimizing the least squares deviation: $$\%\text{apoptosis}={\text{Effect}}_{\max }\ast {[\mathrm{L}-\mathrm{DOPA}]}^{\mathrm{n}}∕{\left({\mathrm{EC}}_{50}\right)}^n+{[\mathrm{L}-\mathrm{DOPA}]}^{\mathrm{n}},\text{where}\mathrm{n}\text{is equivalent to the Hill coefficient}$$ The EC₅₀ values for L-DOPA induced apoptosis calculated from the fitted curves are 49 μM (plus BSO) and 127 μM (minus BSO).

Figure 2. Grx is deactivated within SHSY5Y cells upon L-DOPA treatment

(A) Typical western blot showing Grx content in lysates of SHSY5Y cells, .relative to actin (loading control). (B) Bar Graph displaying densitometric quantification of western blots for Grx content in SH-SY5Y cells under various conditions. Solid bars represent cells with replete GSH. Open bars indicate cells treated with 0.1mM BSO. No statistically significant change in Grx content is evident, n=3, p>0.5 for most comparisons. The content value for 100μM L-DOPA treatment tends toward an anomalous, but insignificant increase in Grx content (n=8, p=0.17). (C) Bar Graph displaying Grx activity assayed according to release of radiolabel ([³H] GSSG) from the prototypical substrate, [³H] BSA-SSG. Solid bars represent cells with replete GSH. Open bars indicate cells treated with 0.1mM BSO. Grx activity for control is 0.854 ± 0.21 nmol/min/mg protein; Grx activity for BSO treated control is 0.703 ± 0.188 nmol/min/mg protein. BSO provided ~2 fold increase in loss of Grx activity. * represent significant difference compared to control, , p<0.01 (n≥3).

Figure 2. Grx is deactivated within SHSY5Y cells upon L-DOPA treatment

(A) Typical western blot showing Grx content in lysates of SHSY5Y cells, .relative to actin (loading control). (B) Bar Graph displaying densitometric quantification of western blots for Grx content in SH-SY5Y cells under various conditions. Solid bars represent cells with replete GSH. Open bars indicate cells treated with 0.1mM BSO. No statistically significant change in Grx content is evident, n=3, p>0.5 for most comparisons. The content value for 100μM L-DOPA treatment tends toward an anomalous, but insignificant increase in Grx content (n=8, p=0.17). (C) Bar Graph displaying Grx activity assayed according to release of radiolabel ([³H] GSSG) from the prototypical substrate, [³H] BSA-SSG. Solid bars represent cells with replete GSH. Open bars indicate cells treated with 0.1mM BSO. Grx activity for control is 0.854 ± 0.21 nmol/min/mg protein; Grx activity for BSO treated control is 0.703 ± 0.188 nmol/min/mg protein. BSO provided ~2 fold increase in loss of Grx activity. * represent significant difference compared to control, , p<0.01 (n≥3).

Figure 3. GR activity is not affected by L-DOPA treatment, however the thioredoxin system proteins, TR and Trx, are diminished upon L-DOPA treatment

(A) Bar graph of relative activities. Activities of the respective enzymes were measured as described under Methods for SHSY5Y cells treated with L-DOPA, either in the absence of BSO (replete GSH, top panel), or in the presence of 0.1mM BSO (decreased GSH, bottom panel). Black bars represent Trx activity; white bars – TR activity; gray bars – GR activity. Upper panel – replete GSH: Trx activity (black bars) showed significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). Trx activity measured for the control is 1.76 ± 0.04 U/mg protein. TR activity (white bars) showed significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). TR activity measured for the control is 1.98 ± 0.32 U/mg protein. GR activity (gray bars) was not significantly changed. GR activity for the control is 2.2 ± 0.3 U/mg protein. Lower panel – diminished GSH: Trx activity (black bars) showed small but significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). Trx activity for the control is 1.91 ± 0.26U/mg protein. Decreases in TR activity (white bars) indicated a 5-fold increased sensitivity to L-DOPA when GSH was diminished. TR activity for the control is 1.92 ± 0.07 U/mg protein. GR activity (gray bars) was not significantly changed. GR activity for the control is 1.81 ± 0.19 U/mg total protein. (B) Typical western blot showing TR and Trx reactivity relative to actin (loading control). (C) Bar graph of relative contents - Densitometric quantification of TR and Trx content using Quantity One software. All samples were normalized to actin, the loading control. * indicates p<0.05, n=3.

Figure 3. GR activity is not affected by L-DOPA treatment, however the thioredoxin system proteins, TR and Trx, are diminished upon L-DOPA treatment

(A) Bar graph of relative activities. Activities of the respective enzymes were measured as described under Methods for SHSY5Y cells treated with L-DOPA, either in the absence of BSO (replete GSH, top panel), or in the presence of 0.1mM BSO (decreased GSH, bottom panel). Black bars represent Trx activity; white bars – TR activity; gray bars – GR activity. Upper panel – replete GSH: Trx activity (black bars) showed significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). Trx activity measured for the control is 1.76 ± 0.04 U/mg protein. TR activity (white bars) showed significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). TR activity measured for the control is 1.98 ± 0.32 U/mg protein. GR activity (gray bars) was not significantly changed. GR activity for the control is 2.2 ± 0.3 U/mg protein. Lower panel – diminished GSH: Trx activity (black bars) showed small but significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). Trx activity for the control is 1.91 ± 0.26U/mg protein. Decreases in TR activity (white bars) indicated a 5-fold increased sensitivity to L-DOPA when GSH was diminished. TR activity for the control is 1.92 ± 0.07 U/mg protein. GR activity (gray bars) was not significantly changed. GR activity for the control is 1.81 ± 0.19 U/mg total protein. (B) Typical western blot showing TR and Trx reactivity relative to actin (loading control). (C) Bar graph of relative contents - Densitometric quantification of TR and Trx content using Quantity One software. All samples were normalized to actin, the loading control. * indicates p<0.05, n=3.

Figure 3. GR activity is not affected by L-DOPA treatment, however the thioredoxin system proteins, TR and Trx, are diminished upon L-DOPA treatment

(A) Bar graph of relative activities. Activities of the respective enzymes were measured as described under Methods for SHSY5Y cells treated with L-DOPA, either in the absence of BSO (replete GSH, top panel), or in the presence of 0.1mM BSO (decreased GSH, bottom panel). Black bars represent Trx activity; white bars – TR activity; gray bars – GR activity. Upper panel – replete GSH: Trx activity (black bars) showed significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). Trx activity measured for the control is 1.76 ± 0.04 U/mg protein. TR activity (white bars) showed significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). TR activity measured for the control is 1.98 ± 0.32 U/mg protein. GR activity (gray bars) was not significantly changed. GR activity for the control is 2.2 ± 0.3 U/mg protein. Lower panel – diminished GSH: Trx activity (black bars) showed small but significant L-DOPA concentration-dependent decreases, * p<0.05, (n=3). Trx activity for the control is 1.91 ± 0.26U/mg protein. Decreases in TR activity (white bars) indicated a 5-fold increased sensitivity to L-DOPA when GSH was diminished. TR activity for the control is 1.92 ± 0.07 U/mg protein. GR activity (gray bars) was not significantly changed. GR activity for the control is 1.81 ± 0.19 U/mg total protein. (B) Typical western blot showing TR and Trx reactivity relative to actin (loading control). (C) Bar graph of relative contents - Densitometric quantification of TR and Trx content using Quantity One software. All samples were normalized to actin, the loading control. * indicates p<0.05, n=3.

Figure 4. Grx is the only thiol disulfide oxidoreductase inactivated by oxidized L-DOPA

(A) Isolated Grx was incubated with L-DOPA, and aliquots of the reaction mixture were with-drawn every 10 minutes and assayed for enzyme activity as described in Methods. Grx activity was decreased in a time and dose-dependent fashion. (B) Semi-log plot of the data shown in (A). Straight lines with increasing negative slopes are indicative of pseudo first order enzyme inactivation. (C) Each isolated enzyme was incubated in the absence or presence of oxidized L-DOPA, and aliquots of the reaction mixture were withdrawn every 20 minutes and assayed for the respective enzyme activities as described under Methods. GR, Trx and TR activities were unaffected, even when tested in separate experiments where the aliquots were withdrawn at 60 minute intervals. Grx was inactivated in an oxidized L-DOPA-dependent manner; * represents significant differences relative to control, p<0.015 (n=3).

Figure 4. Grx is the only thiol disulfide oxidoreductase inactivated by oxidized L-DOPA

(A) Isolated Grx was incubated with L-DOPA, and aliquots of the reaction mixture were with-drawn every 10 minutes and assayed for enzyme activity as described in Methods. Grx activity was decreased in a time and dose-dependent fashion. (B) Semi-log plot of the data shown in (A). Straight lines with increasing negative slopes are indicative of pseudo first order enzyme inactivation. (C) Each isolated enzyme was incubated in the absence or presence of oxidized L-DOPA, and aliquots of the reaction mixture were withdrawn every 20 minutes and assayed for the respective enzyme activities as described under Methods. GR, Trx and TR activities were unaffected, even when tested in separate experiments where the aliquots were withdrawn at 60 minute intervals. Grx was inactivated in an oxidized L-DOPA-dependent manner; * represents significant differences relative to control, p<0.015 (n=3).

Figure 4. Grx is the only thiol disulfide oxidoreductase inactivated by oxidized L-DOPA

(A) Isolated Grx was incubated with L-DOPA, and aliquots of the reaction mixture were with-drawn every 10 minutes and assayed for enzyme activity as described in Methods. Grx activity was decreased in a time and dose-dependent fashion. (B) Semi-log plot of the data shown in (A). Straight lines with increasing negative slopes are indicative of pseudo first order enzyme inactivation. (C) Each isolated enzyme was incubated in the absence or presence of oxidized L-DOPA, and aliquots of the reaction mixture were withdrawn every 20 minutes and assayed for the respective enzyme activities as described under Methods. GR, Trx and TR activities were unaffected, even when tested in separate experiments where the aliquots were withdrawn at 60 minute intervals. Grx was inactivated in an oxidized L-DOPA-dependent manner; * represents significant differences relative to control, p<0.015 (n=3).

Figure 5. Quinone adduction occurs on the active site Cys 22 of Grx

(A). Adduction of 194 Da was seen with the sample treated with oxidized L-DOPA (bottom) compared to the untreated sample (top). (B) MS/MS spectrum of untreated Grx. Fragmentation shows complete coverage of the active site. (C) MS/MS spectrum of L-DOPA treated Grx. Fragmentation shows adduction of 194 m/z to Cys 22 within the active site.

Figure 5. Quinone adduction occurs on the active site Cys 22 of Grx

(A). Adduction of 194 Da was seen with the sample treated with oxidized L-DOPA (bottom) compared to the untreated sample (top). (B) MS/MS spectrum of untreated Grx. Fragmentation shows complete coverage of the active site. (C) MS/MS spectrum of L-DOPA treated Grx. Fragmentation shows adduction of 194 m/z to Cys 22 within the active site.

Figure 5. Quinone adduction occurs on the active site Cys 22 of Grx

(A). Adduction of 194 Da was seen with the sample treated with oxidized L-DOPA (bottom) compared to the untreated sample (top). (B) MS/MS spectrum of untreated Grx. Fragmentation shows complete coverage of the active site. (C) MS/MS spectrum of L-DOPA treated Grx. Fragmentation shows adduction of 194 m/z to Cys 22 within the active site.

Figure 6. Knockdown of Grx results in increased apoptosis in SHSY5Y cells

(A) Typical western blot confirming Grx1 knockdown relative to controls and compared to actin (loading control). (B) Bar graph showing densitometric quantification of Grx1 knockdown. (C) Bar graph showing relative apoptosis for control SHSY5Y cells vs. cells in which Grx1 was knocked down (Grx1-siRNA). Apoptosis was measured in triplicate by chromatin condensation in a blinded fashion using Hoechst 33342 dye. * indicates p<0.01, n=3 independent biological samples.

Figure 7. Inhibition of TR increases apoptosis in SHSY5Y cells

(A) Bar graph showing auranofin concentration-dependent increase in apoptosis of SHSY5Y cells. Apoptosis was measured 24 hr after treatment according to chromatin condensation (Hoechst staining). * indicates p<0.01, n=3; measurements done in triplicate. (B) Bar graph showing auranofin concentration-dependent inhibition of TR in SHSY5Y cells. Auranofin was titrated to give inhibition equal to that seen with L-DOPA treatment. #indicates p<0.05, * indicates p<0.01, n=3.

Figure 7. Inhibition of TR increases apoptosis in SHSY5Y cells

(A) Bar graph showing auranofin concentration-dependent increase in apoptosis of SHSY5Y cells. Apoptosis was measured 24 hr after treatment according to chromatin condensation (Hoechst staining). * indicates p<0.01, n=3; measurements done in triplicate. (B) Bar graph showing auranofin concentration-dependent inhibition of TR in SHSY5Y cells. Auranofin was titrated to give inhibition equal to that seen with L-DOPA treatment. #indicates p<0.05, * indicates p<0.01, n=3.

Scheme 1

A. Selective activities of the thioredoxin (Trx) and glutaredoxin (Grx)systems Grx selectively reduces glutathionylated proteins (Protein-SSG), whereas Trx is able to reduce intermolecular disulfides (Protein-SS-Protein) and intramolecular disulfides (). B. Pro-oxidant metabolism of L-DOPA. L-DOPA is oxidized to semiquinone and quinone forms, releasing superoxide. Also, L-DOPA undergoes a decarboxylation reaction by L-amino acid decarboxylase to form dopamine. Dopamine can undergo similar oxidations to L-DOPA forming both semiquinone and quinone species. Formation of these oxidized products results in the release of superoxide. Lastly, dopamine is degraded by monoamine oxidase forming 3,4-dihydroxyphenylacetic acid (DOPAC). Formation of this degradation product releases superoxide. Also, DOPAC can similarly form the reactive quinone species as shown in the bottom of 1B.

Scheme 1

A. Selective activities of the thioredoxin (Trx) and glutaredoxin (Grx)systems Grx selectively reduces glutathionylated proteins (Protein-SSG), whereas Trx is able to reduce intermolecular disulfides (Protein-SS-Protein) and intramolecular disulfides (). B. Pro-oxidant metabolism of L-DOPA. L-DOPA is oxidized to semiquinone and quinone forms, releasing superoxide. Also, L-DOPA undergoes a decarboxylation reaction by L-amino acid decarboxylase to form dopamine. Dopamine can undergo similar oxidations to L-DOPA forming both semiquinone and quinone species. Formation of these oxidized products results in the release of superoxide. Lastly, dopamine is degraded by monoamine oxidase forming 3,4-dihydroxyphenylacetic acid (DOPAC). Formation of this degradation product releases superoxide. Also, DOPAC can similarly form the reactive quinone species as shown in the bottom of 1B.