Effects of Hydrogen Sulfide-releasing l-DOPA Derivatives on Glial Activation

Introduction

The pathogenesis of Parkinson disease (PD)² results primarily from loss of neurons, especially dopaminergic neurons of the 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 disease 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 activated microglia and astrocytes (7–10). Hybrid molecules which combine l-DOPA with antioxidant and antiinflammatory moieties might therefore have therapeutic potential.

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 β-synthase (CBS) and cystathione γ-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 molecule because it has multiple physiological and pathophysiological functions in body organs. A protective role for H₂S in neurons against oxidative stress has been reported (14). It has also been demonstrated that H₂S induces alterations in calcium channels [Ca²⁺]_i in 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, protective 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 demonstrated in vivo in the 6-OHDA rat model of Parkinson disease (20).

To date there are no disease-modifying drugs for the treatment of PD. As an approach to the development of next generation agents, we have prepared hybrid compounds designed to combine the dopamine replacement properties of l-DOPA with the neuroprotective properties of H₂S donors.

In this investigation, we synthesized four H₂S-releasing moieties (ACS48, ACS50, ACS5, and ACS81) and examined whether they release H₂S or equivalent SH⁻ ions in both glia and neurons. The sulfurated moieties were chosen among those resembling the structures of known H₂S-releasing compounds such as the dithiolethione ADT-OH (21) and diallyldisulfide (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.

EXPERIMENTAL PROCEDURES

RESULTS

First, we evaluated the release rate in vivo of the H₂S donor from ACS84, a typical l-DOPA hybrid compound, administered 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). Measurements performed at earlier times from the treatments indicate that demethylation of ACS84 occurs rapidly, whereas the cleavage of the amide bond between dithiolethione and l-DOPA occurs more slowly (data not shown). Low micromolar or submicromolar concentrations of ACS84 and its main metabolites were found in brain 1 h after administration.

Dopamine levels in brain were increased 2.2-fold by treatment with ACS84. Interestingly, GSH, a known antioxidant and neuroprotective agent (32) was increased 1.4-fold by this treatment. 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.

We commenced by examining the rate of cleavage of the H₂S-releasing moieties (ADT-OH, ACS48, ACS50, ACS5, and ACS81) under extracellular conditions. We tested 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 supplemental 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 μm 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).

These data establish that all the moieties are metabolized within each cell type to generate H₂S. To explore a possible mechanism, we purified functional mitochondria from U373 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 (−)-deprenyl, a classical inhibitor of monoamine oxidase B, were metabolized 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.

Within cells, the most important reducing agent is glutathione (GSH). To determine whether the H₂S donors were affecting GSH levels, we measured [GSH]_i in SH-SY5Y cells exposed to the four different S-DOPAs (10 μm 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]_i in SH-SY5Y cells under normal conditions by ∼1.5-fold (p < 0.01). We then exposed the cells for 1 day to conditioned medium from stimulated THP-1 cells (LPS/IFNγ for 2 days) or U373 cells (IFNγ for 2 days). This treatment caused a huge decrease in [GSH]_i (∼90% decrease, p < 0.01). NaSH and the four H₂S-releasing S-DOPAs significantly attenuated this decrease (p < 0.01). Nevertheless, the values were still significantly lower than those obtained from control media. These data establish that H₂S generated from the donor compounds is significantly converted into the antioxidant [GSH]_i and that this [GSH]_i is 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 μm 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 < 0.01) after exposure of SH-SY5Y cells to media from unstimulated THP-1 or U373 cells and about 65% after exposure to medium from THP-1 or U373 cells stimulated for 24 h (p < 0.01). These data establish that all the S-DOPAs are selective MAO B inhibitors.

We next investigated the effects of 1, 3, 10, 30, and 50 μm 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, (−)-deprenyl, ACS83, ACS84, ACS85, or ACS86 to LPS/IFNγ-activated THP-1 cells (5A) or IFNγ-activated U373 cells (5B) at a standard concentration of 10 μm. It was found that the H₂S-releasing agents ADT-OH and NaSH, but not l-DOPA or (−)-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 μm, and 2, 4, 8, and 12 h pretreatment are shown in supplemental Figs. S4 and S5. Again, the results were highly similar for all the SH-donors and all the S-DOPA derivatives in their concentration and incubation time dependence.

The 8-h time period shown in Fig. 5 demonstrated a robust effect of THP-1 and U373 cells, so it was deemed optimum for showing comparative effects using human microglia and astrocytes. It was found that the protective effect of pretreatment with NaSH and the S-DOPAs (10 μm, 8 h) was comparable in human microglia and astrocytes to that found for THP-1 and U373 cells (Fig. 6A: microglia and 6B: astrocytes). MTT data are shown in the upper panels and LDH release data in the lower panels. More detailed experimental data (3, 10, and 50 μm; 8 h of preincubation) showing comparable effects at differing concentrations are shown in supplemental Fig. S6.

Inflammatory stimulation of microglia or THP-1 cells causes them to release the inflammatory cytokines TNFα and IL-6, as well as to generate neurotoxic nitrite ions. Fig. 7 shows the effect of treatment with NaSH, ADT-OH, and the four S-DOPA compounds (10 μm each, 8 h of preincubation) on THP-1 release of TNFα (Fig. 7A), IL-6 (Fig. 7C), and nitrite ions (Fig. 7E), and on human microglial release of TNFα (Fig. 7B), IL-6 (Fig. 7D) and nitrite ions (Fig. 7F). There was a substantially lower release of nitrite ions by stimulated microglia compared with THP-1 cells, even allowing for the 10-fold lower number of microglial cells that were seeded. This is perhaps related to the reported poor ability of human microglia to express iNOS. Nevertheless there were significant differences between the release of these materials in both types of cells: (1) between stimulated 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 compounds (p < 0.01). However, l-DOPA or (−)-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 IFNγ as described under “Experimental Procedures” and were then treated similarly to the THP-1 and microglial 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 stimulated cells that were untreated (p < 0.01). Complete data showing a similar concentration dependence of the four donor moieties and the four S-DOPA derivatives on H₂S release (3, 10, and 50 μm; 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-donating 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 + 84, ACS 84 + 85, ACS 84 + 86) compared with the same derivatives alone.

DISCUSSION

In the present study, we examined the antioxidant and antiinflammatory 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 astrocytes, as well as the humanTHP-1 U373 cell lines and then generating intracellular H₂S. The H₂S they generated acted in part to enhance levels of the classical antioxidant GSH. They also inhibited MAO B. Moreover, they acted as antiinflammatory compounds by ameliorating the neurotoxic effects of glial cell supernatants toward SH-SY5Y cells. Additionally, they inhibited release of the proinflammatory mediators TNFα, IL-6, and nitrite ions (Figs. 7 and 8). The compounds were equipotent, but l-DOPA was without effect in all of these assays.

We have previously reported that depleting intracellular GSH by inhibiting its synthetic enzyme γ-glutamylcysteine synthase induces an inflammatory reaction in glial cells with consequent 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 demonstrates that a significant amount of the intact, and very lipophilic ACS84, crosses the blood brain barrier and is hydrolyzed in the brain into its two components l-DOPA and ACS50. Moreover the concomitant inhibition of the dopamine-metabolizing enzyme MAO B, as had previously been shown for dithiolethiones (33), could contribute to sustain the high concentration of dopamine in brain. The increase of GSH presumably results from the H₂S generated by metabolism of the donor moiety. While very preliminary, these data demonstrate 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 glutathione (14) thereby inhibiting oxidative 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 from oxidized l-DOPA compounds.

As far as neuroinflammation is concerned, it is well known that areas affected in PD, especially the substantia nigra, are characterized by the presence of activated microglia and activated astrocytes (4, 7–10, 35, 36). There is comparable glial activation in animal models of PD (37). Evidence that these reactive glial cells contribute to the neuronal degeneration comes from epidemiological studies where persons taking NSAIDs are reported to be relatively spared from PD (38, 39) especially if combined with coffee (40). Such protection is also reported for animal models of PD (37, 41).

The MPTP phenomena may be particularly revealing with respect to the potential consequences of inducing SN inflammation. Drug addicts who were originally exposed to MPTP developed a relentlessly progressive parkinsonian syndrome. Postmortem studies showed persistent inflammation up to 17 years following their last exposure to the toxin (35). This human experience was duplicated in monkeys where inflammation of the SN was demonstrated up to 14 years after their last MPTP exposure (8). This suggests that inflammation of the SN, once initiated, may be self sustaining. If this is the case, antiinflammatory treatment may be essential to arresting progression of PD.

Our study demonstrates that inflammatory stimulation of glial cells results in a 5–7-fold decrease in intracellular H₂S (see supplemental Fig. S11). This indicates an increase in intracellular consumption as reactive processes are induced. These experiments were performed in the presence of hydroxylamine to suppress endogenous H₂S synthesis by CBS. But in a previous study (12) we showed that such inflammatory stimulation also sharply reduced the expression of CBS. Therefore inflammatory stimulation reduces intra-glial production of H₂S while at the same time increasing consumption. The result is a deprivation of this endogenous anti-inflammatory and neuroprotective agent.

There are studies demonstrating that serum levels of homocysteine, an intermediate of the trans-sulfuration pathway, were increased in PD patients receiving l-DOPA (42–44). One of the main enzymes responsible for hyperhomocysteinemia is CBS, which is mainly expressed in astrocytes in brain (11, 12). So it could be hypothesized that endogenous production of H₂S is reduced in PD brain, thus compounding the potential damage of oxidative stress associated with l-DOPA administration. This provides additional rationale for designing methods of supplementing H₂S availability in PD.

We have previously shown that SH-SY5Y cells express both MAO A and B (45). MAO B is primarily responsible for oxidative degradation of dopamine (46, 47). Treatment with NaSH and H₂S-releasing moieties selectively inhibited MAO B (Fig. 4C). This could be one of the reasons why ACS84-treated rats have higher dopamine levels than vehicle or l-DOPA injected rats (Table 2). However, we could not exclude that dopamine is generated from degradation of ACS84 in rats. MAO B activity in SH-SY5Y cells was less inhibited in cells exposed to the inflammatory effects of THP-1- or U373 cell-conditioned medium. This could be due to consumption of the generated H₂S in stimulated cells, presumably through oxidative reactions.

In conclusion, our data demonstrate that H₂S not only has anti-inflammatory activity against the glial toxicity which may be associated with the pathogenesis of PD (36), but may also reduce the stress induced by l-DOPA oxidation (14). Furthermore, by inhibiting MAO B, H₂S-releasing l-DOPA compounds could help restore the disease-depleted dopamine levels. The H₂S-releasing l-DOPA derivatives (S-DOPAs) described here represent compounds that can reach the brain and, in cells under stress, deliver ameliorating SH⁻ ions in a time-dependent manner. They have properties that make them candidates for future treatment of PD. However the long term consequences of their use will require much further study.

Supplementary Material