Neurotrophic Effects of l-DOPA in Postnatal ... - Wiley Online Library

Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry

Neurotrophic Effects of L-DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures *~M~j.ja Angeles Mena, *Viviana Davila, and *David Suizer *

Departments of Neurology and Psychiatry, Columbia University and Department of Neuroscience, New York State Psychiatric Institute, New York, New York, U.S.A.; and ~Departamento de Investigación, Hospital Ramón y Cajal, Madrid, Spain

Abstract: L-DOPA IS toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of L-DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum-free, glia-conditioned medium. L-DOPA (50 tiM) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia-free cultures, where L-DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 1iM LDOPA. The stereoisomer D-DOPA (50—400 btM) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 ~zM)did not block the neurotrophic effect. These data suggest that the neurotrophic effect of L-DOPA is stereospecific but independent of the production of dopamine. However, L-DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by Lbuthionine sulfoximine (3 1iM for 24 h) blocked the neurotrophic action of L-DOPA. N-Acetyl-L-cysteine (250 1iM for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of L-DOPA. These data suggest that the neurotrophic effect of L-DOPA may be mediated, at least in part, by elevation of glutathione content. Key Words: L-DOPA—Glia—Glutathione—Parkinson‘s disease—Dopamine neurons. J. Neurochem. 69, 1398—1408 (1997).

The effects of L-DOPA on dopamine (DA) neurons are quite different in vivo and in vitro. Whereas relatively low levels (25 jiM) of L-DOPA are toxic in culture (Melamed, 1986; Mena et al., 1992, 1993; Mytilineou et al., 1993; Pardo et al., 1995), the drug has not been seen to damage DA neurons in healthy animals (Hefti et al., 1981; Perry et al., 1984; Yurek et al., 1991; Blunt et al., 1993) or humans (Quinn et al., 1986). As L-DOPA is the most effective compound for symptomatic treatment of Parkinson‘ s disease (PD), its influence on the progression of the disease is particularly important. Although some reports have indicated increased fluctuations and dyskinesia related to the duration of the L-DOPA treatment (Lesser et al., 1979), other studies have found no evidence of

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negative impact (Markham and Diamond, 1986; Blint et al., 1988; Uitti et al., 1993). Previous studies on L-DOPA toxicity in culture have used embryonically derived cells grown in the presence of a very low level of glia ( 100 ‚aM, but there was no neurotoxicity

TABLE 1. Effect of L-DOPA (48 h) on neuronal survival and quinone formation Cells/cm

Controls L-DOPA 25 pM

50 pM 100 pM 200 pM 400 pM

2 Quinones

TH-positive

TH-negative

% TH-positive

645 ±34 (100%)

1,251 ± 123 (100%)

34.2 ± 1.54

369 ± 19 (100%)

1,130 1,076 1,451 1,426 919

±88 (90%) ±75 (86%) ±10 (116%)

45.3 ±3.6° 49.8 ± l.3l~ 34.8 ±0.92

± 11

(114%)

33.2 ± 1.3

±87° (73.4%)

36.6 ±3.1

367 ± 15 (100%) 328 ± II (89%) 343 ± 11 (93%) 373 ± 11 (101%) 635 ±26~(172%)

949 1,071

±516 (147%) ±58~(166%) 774 ±65 (120%) 710 ±71 (110%)

534 ±49 (82.8%)

(OD X 10~)

Data are mean ± SEM values. Numbers of TH-positive and -negative cells/cm2 are from two to six experiments (n > 6 for each data point). Cultures were treated with L-DOPA for 48 h after 5 days in culture. °p < 0.05,

J. Neurochem., Vol. 69, No. 4, 1997

b~
80 pm from a point in the center of the cell body and increased the number of TH-positive neurons that displayed more than three primary processes. Data are expressed as mean ±SEM (bars) percentages of TH-positive neurons that displayed more than three primary processes versus total 1H-positive neurons (% TH + MP) and as mean ±SEM (bars) percentages of TH-positive neurons with processes >80 pm versus total TH-positive neurons (% TH + LP). *p 50% by L-DOPA. This

may provide an explanation for discrepancies between the results of L-DOPA in the various models. The neurotrophic actions of L-DOPA may underlie the longterm improved motor control after the cessation of drug administration. Previous studies indicate that DA added to culture medium increases the expression of TH and aromatic amino acid decarboxylase in primary cultures of fetal neurons (De Vitry et al., 1991), and the neurotransmit-

ter itself has been postulated as a neurotrophic factor (Leslie, 1993). Normally, L-DOPA in neuronal culture is rapidly converted to DA and taken up into synaptic vesicles (Pothos et al., 1996). If conversion of LDOPA to DA is required for its neurotrophic effects, the response should be blocked by the aromatic acid decarboxylase inhibitor carbidopa (Basma et al., 1995). Our study reveals therefore that the mechanism responsible for the neurotrophic effect of L-DOPA is independent of its conversion to DA.

FIG. 5. Comparison of the effect of long-term

L- and D-DOPA on TH-positive neurons. Micrographs show TH immunostaining of postnatal ventral midbrain cells treated with L-DOPA (200 pM) or D-DOPA (200 pM). Cells were treated at 5 days postplating for 8 days with (A) vehicle, (B) L-DOPA (200 pM), or (C) o-DOPA (200 pM). Bar = 30 pm.

the total glutathione (Pan and Perez-Polo, 1993; Ferrari et al., 1995), and it was previously found that LDOPA-induced elevated glutathione levels in midbrain cultures were due predominantly to an increased GSH content (Mytilineou et al., 1993). We found that 50 ‚aM L-DOPA increased GSH content to 137% of control levels in midbrain neurons/cortical glia cocultures (from 20.8 ± 1.3 to 28.6 ± 2.2 nmol/mg of protein; n = 8, two experiments; p < 0.01).


Astrocytes produce several neurotrophic and neurite-promoting agents (Engele et al., 1991; Nagata et al., 1993; Hoffen et al., 1994; Takeshima et al., 1994; Muller et al., 1995) that influence development, survival, neunte extension, and neurotoxin resistance (Engele et al., 1991; Nagata et al., 1993; Takeshima et al., 1994). Among the systems identified are a glutamate uptake activity that protects against excitotoxicity (Rosenberg et al., 1991) and stimulation of GSH syn-

thesis and enzymes involved in GSH metabolism (Nisticè et al., 1992). GSH plays a major role in protection against oxidative stress (Meister, 1988; Makar et al., 1994) and functions to remove the H 202 generated by monoamine oxidase (Werner and Cohen, 1993). GSH is produced by GSH peroxidase in culture (Raps et al., 1989). GSH peroxidase is mainly a mitochondrial enzyme (Victonica et al., 1984) exclusively located in

the human midbrain within glial cells (Slivka et al., 1987; Damier et al., 1993). GSH levels decrease with age in neurons but remain stable in astrocytes in culture (Sagara et al., 1996). However, neurons can maintain an intracellular glutathione poo1 by taking up cysteine provided by glial cells (Sagara et al., 1993).

L-DOPA AND POSTNATAL DOPAMINE COCULTURE

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FIG. 6. Effects of L-NAC and GSH on postnatal ventral midbrain cells. Cultures were treated at 5 days postplating for 48 h with (A) vehicle, (B) L-NAC (0.25 mM), (C) L-NAC (0.5 mM), (D) L-NAC (1 mM), (E) GSH (200 pM), or (F) GSH (400 pM). Bar = 30 pm.

Protection from oxidation enhances the survival of mesencephalic cultures (Mena et al., 1993, 1996; Pardo et al., 1995), and the number of TH-positive neurons is increased if SOD, USH peroxidase, or LNAC is added to the medium (Colton et al., 1995). We have shown that TH-positive neurons that overexpress SOD display increased neutite outgrowth and survival (Przedborski et al., 1996) and are protected from LDOPA toxicity (Mena et al., 1997). Recently, Han et al. (1996) have reported that L-DOPA raises GSH levels in cultures of fetal rat mesencephalon, a mouse neuroblastoma line (Neuro-2A), a human neuroblastoma (SKNMC), and glia from newborn rat brain but not in midbrain neuronal cultures grown without glia.

In the present study, the effects of L-DOPA were not associated with an elevation in content of extracellular quinones, in contrast with the observations in embryonically derived, glial-free cultures (Mena et al., 1996), suggesting that astrocytes may inhibit L-DOPA quinone formation by providing an antioxidant system. D-DOPA was not neurotrophic, although it was more effective in inducing formation of extracellular quinones. Moreover, we found that the GSH synthesis promoter L-NAC and GSH itself reproduced L-DO-

PA‘s effects, whereas the GSH synthase inhibitor BSO blocked L-DOPA‘ s effect. Therefore, the present study suggests that L-DOPA is neurotrophic owing to stimulation of GSH peroxidase. It is possible that L-DOPA is sequestered intracellularly in glia by L-amino acid


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M. A. MENA ET AL.

erer et al., 1989; Sofic et al., 1992; Sian et al., 1994). L-DOPA treatment reverses GSH depletion (Tohgi et al., 1995). In normal subjects, the nigrostriatal DA neurons, the most important disease target associated

TABLE 5. Effect of L-NAC and GSH (48 h) on neuronal survival Cells/cm TH-positive Controls L-NAC 1 mM 0.5 mM 0.25 mM GSH 200 pM 400 pM

2 %

TH-negative

342 ±17 (100%)

919 ±46 (100%)

27.3 ±2.1 26.2 ±3.1

623 ±49“ (182%)

1,030 ±97 (111%) 1,119 ±203 (121%) 1,061 ±97 (115%)

539 ±16“ (157%) 603 ±62‘ (176%)

1,026 ± 106 (112%) 946 ±92 (103%)

34.1 ± 1.9“ 39.1 ± 1.1‘

365 ±42

(107%)

500 ±46“ (146%)

with PD, are normally surrounded by a low density of glia (Damier et al., 1993; Sagara et al., 1993; Makar et al., 1994), suggesting that these neurons are less protected from oxidative stress. It is striking that, in PD, the density of GSH peroxidase-positive glia surrounding the surviving DA neurons is increased, suggesting a compensatory proliferation of glia that protects surviving neurons against pathological death (Damier et al., 1993).

TH-positive

31.2 ±3.2 37.2 ±3.1“

Although L-DOPA is toxic for catecholamine neurons in other in vitro systems (Melamed, 1986; Mena et aI., 1992, 1993; Pardo et al., 1995), catecholamines

Data are mean ±SEM values. Numbers of TH-positive and -negative cells/cm2 are from two experiments (n = 10 foreach data point). Cultures were treated with L-NAC or GSH for 48 h after 5 days in culture, “p < 0.05, h~ < 0.01, ‘p < 0.001 versus controls.

are required for the development of the nervous system and survival. Inactivation of the TH gene produced the death of 90% of mutant mice fetuses, whereas administration of L-DOPA to pregnant females resulted in complete rescue of mutant mice (Zhou et al., 1995).

transport systems and in turn stimulates astrocytic GSH

peroxidase.

Transgenic animals with mutations of the TH gene

It appears unlikely that L-DOPA provides its neurotrophic effect by acting as an antioxidant itself because

have a poorer prognosis than those with mutations of the dopamine /3-hydroxylase gene (Thomas et al., 1995), suggesting that DA is essential for development of the nervous system. It is likely that L-DOPA and catecholamines are critical factors for promoting the survival and differentiation of DA neurons and that the effects of L-DOPA on DA cells in vivo might be neurotrophic or neurotoxic depending on the local environment produced by glial cells.

high doses of L-DOPA do not provide neuroprotection and increase quinone formation; however, a cautionary note is provided by the finding that L-NAC, previously

assumed to act as an antioxidant due to its role as a substrate for GSH synthesis, may protect PC12 cells by acting directly as an oxygen radical scavenger (Yan et aI., 1995). L-NAC is a pluripotent protector that elevates the level of intracellular glutathione, prevents

against apoptotic death of neuronal cells, and enhances

In summary, conclusions from this study and that

trophic factor-mediated cell survival (Mayer and Noble, 1994; Ferrari et al., 1995). Indeed, depletion of brain GSH with BSO in rats is accompanied by impaired mitochondrial function and decreased complex IV activity (Heales et al., 1995) leading to energy crisis as a mechanism of nigral cell death (Jenner,

of Mena et al. (1996) indicate that a long-term potentiation of DA release by L-DOPA may occur owing to promotion of neunitic arbonization and protection

1993). In clinical studies, GSH content is decreased in the substantia nigra of patients with idiopathic PD (Ried-

mechanisms such as up-regulation of GSH. Significant

against cell death. The neurotrophic effects are independent of conversion to DA and dependent on the presence of astrocytes and may result from antioxidant questions that need to be addressed include whether the effect is due to L-DOPA itself acting as an antioxi-

TABLE 6. Effect of L-DOPA and BSO (48 h) on neuronal survival and quinone formation Cells/cm2 TH-positive Controls L-DOPA (50 pM) BSO (3 pM) L-DOPA + BSO

450 ±18 720 ±72“ 436 ±17 441 ±40“

(100%) (160%) (96.8%) (98%)

TH-negative 1,303 1,408 1,160 1,329

±60 ±143 ±72 ±11

(100%) (108%) (89%) (102%)

Quinones (OD X l0~)

% TH-positive 25.7 33.9 26.7 24.6

±2.1 ±0.8~‘ ±1.05 ±0.8

285 284 293 276

±1.8 ±7 ±3 ±4

(100%) (99.7%) (103%) (96.7%)

Data are mean ±SEM values. Numbers of TH-positive and -negative cells/cm2 are from two experiments (n = 10 for each data point). Cultures were treated after 5 days in vitro with 50 pM L-DOPA (for 48 h) or 3 pM L-BSO alone or in combination. Pretreatment with BSO for 24 h was followed by washout with neuronal medium, before L-DOPA or solvent was added. b~ < 0.01 versus controls. “p < 0.001 for cultures treated with L-DOPA + BSO versus L-DOPA.


L-DOPA AND POSTNATAL DOPAMINE COCULTURE dant or if it is due to up-regulation of a cellular system such as the GSH peroxidase pathway, whether sequestration of DA by synaptic vesicles plays a role in protection from L-DOPA toxicity, and the precise pathways of exchange of cysteine or other protective mechanisms between glial cells and DA neurons.

Acknowledgment: This work was funded by the Parkinson‘s Disease Foundation, grants 10154 and 07418 from the National Institute on Drug Abuse (to D.S.), and grants PR 95-098 and SAF 96-1099 from the Spanish MEC. Our thanks to Johanna Bogulavsky and Irma Ryjak for expert technical assistance and to Mrs. Liselotte Gulliksen for editorial help.

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