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Melatonin rescues dopamine neurons from cell death in tissue culture models of oxidative stress
Brain Research 768 Ž1997. 317–326
Research report
Melatonin rescues dopamine neurons from cell death in tissue culture models of oxidative stress Lorraine Iacovitti ) , Natalie D. Stull, Kelly Johnston Department of Neurobiology and Anatomy, Mailstop 408, Allegheny UniÕersity of the Health Sciences, Broad and Vine Streets, Philadelphia, PA 19102, USA Accepted 13 May 1997
Abstract Dopamine ŽDA. neurons are uniquely vulnerable to damage and disease. Their loss in humans is associated with diseases of the aged, most notably, Parkinson’s Disease ŽPD.. There is now a great deal of evidence to suggest that the destruction of DA neurons in PD involves the accumulation of harmful oxygen free radicals. Since the antioxidant hormone, melatonin, is one of the most potent endogenous scavengers of these toxic radicals, we tested its ability to rescue DA neurons from damagerdeath in several laboratory models associated with oxidative stress. In the first model, cells were grown in low density on serum-free media. Under these conditions, nearly all cells died, presumably due to the lack of essential growth factors. Treatment with 250 mM melatonin rescued nearly all dying cells Ž100% tauq neurons., including tyrosine hydroxylase immunopositive DA neurons, for at least 7 days following growth factor deprivation. This effect was dose and time dependent and was mimicked by other antioxidants such as 2-iodomelatonin and vitamin E. Similarly, in the second model of oxidative stress, 250 mM melatonn produced a near total recovery from the usual 50% loss of DA neurons caused by neurotoxic injury from 2.5 mM 1-methyl-4-phenylpyridine ŽMPPq.. These results indicate that melatonin possesses the remarkable ability to rescue DA neurons from cell death in several experimental paradigms associated with oxidative stress. q 1997 Elsevier Science B.V. Keywords: Melatonin; Oxidative stress; Dopamine neuron; Tissue culture; MPPq; Oxyradical
1. Introduction Almost a century has passed since it was first recognized that Parkinson’s Disease ŽPD. involved the selective loss of neurons from specific nuclei in the ventral midbrain region w49x. Some forty years later, Ehringer and Hornykiewicz Ž1960. w11x demonstrated that the affected cells used dopamine ŽDA. as their neurotransmitter. The most severe loss of DA neurons occurs in the substantia nigra ŽSN.; with more moderate losses in the ventral tegmental area and A8 neurons w20x. Although a number of different theories have been advanced over the years to explain the selective vulnerability of DA neurons in PD, the mechanism of cell destruction and the cause of the disease remain a mystery. One intriguing theory holds that there is cytotoxic loss of DA neurons following oxidative stress to the cells caused by the formation of hydrogen peroxide ŽH 2 O 2 . and ) Corresponding author. Fax: q1 Ž215. 762-3127; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 6 8 - 9
oxygen free radicals Žfor review; w12x.. Indeed, there is an impressive body of evidence to suggest that DA neurons of the SN are at greater risk for oxidative stress and more susceptible to its ravages than other structures in the brain. Firstly, neurons of the SN contain neuromelanin, an autooxidative byproduct of DA metabolism w14x which can lead to the formation of superoxides. Under normal conditions, these oxyradicals are de-toxified by cytosolic CurZn superoxide dismutase ŽSOD.. The fact that SOD mRNA is present in higher than normal levels in PD is suggestive of an increased requirement for superoxide scavengers in diseased DA neurons w4x. Moreover, since SOD itself leads to the production of H 2 O 2 , its increased activity in PD is likely to further exacerbate the stress placed on these cells w35x. Contributing further to their risk of oxidative crisis, is the fact that the normal antioxidative defense system Žcatalase and glutathione peroxidase. used to de-toxify H 2 O 2 is reduced in PD w12x and in the aged brain w7x; resulting in an accumulation of highly toxic hydroxyl radicals. Likewise, iron, which also participates in the formation of hydroxyl radicals from H 2 O 2 , is increased in
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DA neurons in PD w8,10x. These toxic species, in turn, cause lipid peroxidation, provoking disturbances in cell membranes, calcium influx, disruption of the cytoskeleton and a cascade of events leading to cell death w20x. Experimental paradigms mimicking these events in culture make possible the search for substances of potential importance in neuronal salvation from oxidative damage andror death. The first of these models is based upon the belief that, during development, neurons compete for trophic substances found in target tissues. Because these factors are available in limited supply, many neurons are unsuccessful in their bid and consequently initiate a suicide program resulting in their demise. Developmental cell death can be modeled in vitro by depriving cells in culture of these same essential factors. Under these conditions, the general health of cells declines rapidly, resulting in apoptosis Žincluding cell shrinkage, nuclear condensation, DNA fragmentation, blebbing of the plasma membrane. within approximately 48 h wfor review, w26xx. The evidence that oxidative stress underlies developmental apoptosis from growth factor deprivation derives from an elegant series of experiments by Johnson and colleagues. These studies established that the overexpression of SOD w15x or the bcl-2 gene product w13,16x, both of which are antioxidants w6,21,22,27,39,40x, prevents the apoptosis that peripheral neurons undergo following nerve growth factor withdrawal. Although the trigger for cell destruction is quite different from trophic factor deprivation, treatment with the specific CA neurotoxin MPPq w19x represents a second important experimental paradigm for evaluating a substance’s ability to rescue neurons from oxidative cell death. MPPq is thought to exert its toxic effects by impairing complex I of the mitochodrial respiratory chain w42x which induces increased amounts of superoxide radicals, lipid peroxidation w12x homeostatic changes in Ca2q w28,38x and, finally, apoptosis w31x. It is not surprising, in light of the important role that oxygen free radicals play in producing these toxic effects Žfor review, Fahn and Cohen, 1992 w12x., that the use of antioxidants has become an important new line of investigation. Indeed, several antioxidants Žvitamins A and E. can, with varying degrees of success, protect nigral DA neurons from neurotoxin-induced damage w5,51x. Arguably, the most potent and versatile of cellular antioxidants is the pineal hormone, melatonin Ž5-methoxyN-acetyl-tryptamine., which possesses multiple ways to both reduce free radical production and to neutralize radicals once generated w34x. These effects are accomplished without receptors w30x, presumably because of the indole’s high lipid solubility which allows it free access to the interior of the cell. Typical of a hormone, and in contrast to vitamin antioxidants which are compartmentalized, melatonin is capable of acting in the membrane, cytosol and nucleus w30x. Studies in vitro have demonstrated a remarkable capacity for melatonin to scavenge hydroxyl
w45,46x and peroxyl radicals w33x. Furthermore, melatonin stimulates the antioxidant enzyme, glutathione peroxidase w1x while inhibiting nitric oxide synthase from generating more free radicals w36x. Although virtually nothing is known regarding melatonin’s effects on the DA system, its extraordinary antioxidant properties suggested to us a potential role for melatonin in the rescue of DA neurons damaged by oxidative stress. We will show that melatonin treatment can dramatically salvage DA neurons from the inevitable cell death caused by oxidative stress due to growth factor deprivation or MPPq treatment. 2. Materials and methods 2.1. Tissue culture Pregnant Sprague-Dawley rats were purchased from Taconic Laboratories at 15 days gestation q12 h Žfertilization day s embryonic day 0.. Pregnant dams were deeply anaesthetized with 40 mgrkg sodium pentobarbital while embryos were removed and transferred into Leibowitz-15 medium ŽL-15. for dissection. The developing ventral mesencephalon Ž1 mm2 . was isolated from the remainder of the brain as described previously w25x. After removal of the meninges, tissue was minced and incubated in Ca2q-, Mg 2q-free Hank’s balanced salt solution ŽCMF-HBSS. for 8 min at 378C in a clinical rotator Ž40 rpm.. The incubation mixture was replaced with a 0.01% trypsin solution Žin CMF-HBSS., and incubated for an additional 8 min, rinsed twice in Leibovitz medium ŽL-15., and placed in culture medium containing Dulbecco’s minimum essential medium ŽDMEM., 10% fetal calf serum, glucose Ž6 mgrml., glutamine Ž204 mgrml., and penicillinrstreptomycin Ž100 Urml.. Cells were dissociated by trituration through a reduced bore glass pipette and plated onto plastic Lab Tek 8-well culture slides coated with polymerized polyornithine Žprecoated overnight at 378C at a concentration of 0.01 mgrml in 15 mM borate buffer, pH 8.4. then rinsed with water and air-dried 5 min. The cells were plated at two different densities; high Ž12500 cellsrmm2 . and low Ž500 cellsrmm2 .. After a 1–2 h stabilization period in standard media, cultures were transferred to a chemically defined serum-free media ŽDM. containing 50% DMEM, 50% Ham’s F12 media, 1% ITSq, and glucose, glutamine and penicillinrstreptomycin as described above. 2.2. Cell Õiability and cytotoxicity assays Cell viability and cytotoxicity were assessed based on the principle that live cells contain ubiquitous intracellular esterase activity which can be detected by the enzymatic conversion of the nonfluorescent cell permeant calcein AM to the highly fluorescent calcein Žgreen fluorescence at 530 nm.. Dead cells were detected after ethydium homodimer entered cells through damaged membranes to bind to
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DNA, making them fluoresce red Ž) 600 nm.. Essentially cultured cells were rinsed in PBS, 100 ml of combined LIVErDEAD assay reagents Žfinal concentration 0.5 mM calcein AM and ethydium homodimer. were added and incubated for 45 min at room temperature. Cultures were then viewed on a fluorescence microscope. Apoptosis was assessed using the DNA dye Hoescht 33,258; unhealthy apoptotic cells contain clumped and fragmented DNA while healthy cells do not. 2.3. Immunocytochemistry Cultures were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4. and processed with antibodies to TH Ž1 : 5000 dilution., glial fibrillary acidic protein ŽGFAP. Ž1 : 400 made in mouse. or Tau Ž1 : 200 made in rabbit. using the immunoperoxidaserABC method of staining ŽElite Vectakit. as adapted for tissue culture w24x. The number of immunoreactive neurons was determined by counting positively stained cells in all microscopic fields on the culture dish. This was accomplished with the aid of an eyepiece reticule used at a 10 = magnification.
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ferences were considered significant only when the P value was less than 0.05. 2.5. Chemical reagents GFAP and Tau antibodies were purchased from Sigma Chemical ŽSt. Louis, MO, USA.. TH antibodies were a kind gift of Dr T.H. Joh, New York, NY, USA. All reagents used in culture were obtained from the Gibco Co. or Fisher Scientific with the exception of ITSq purchased from Collaborative Biomedical Products and fetal calf serum from Irvine Scientific. Melatonin ŽCat. a 5250. was purchased from Sigma Chemical ŽSt. Louis, MO, USA.; 2-iodomelatonin ŽCat.a 550-153-M005. from Alexis Corp.; 2-iodo-N-butanoyl-5-methoxytryptamine from RBI ŽCat. a M-112. and the water soluble vitamin E analog, Trolox Ž6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid. from Aldrich Co. Liverdead Cell-mediated Cytotoxicity Kit was purchased from Molecular Probes Inc. ŽEugene, OR, USA..
3. Results 2.4. Statistical analysis 3.1. Effects of melatonin on the deÕelopmental model Data were statistically analyzed by one way analysis of variance. When P - 0.05, then the F-test was followed by the two-tailed Student’s t-test to compare the statistical significance between control and experimental groups. Dif-
In general, neurons flourish in culture even in the absence of exogenous growth factors if they are plated at a sufficiently high density. Presumably the trophic factors
Fig. 1. Photomicrograph of cells from the E15 rat ventral mesencephalon plated at low density Ž500 cellsrmm2 . and maintained for 3–4 days in vitro on DM ŽA,B. or DM supplemented with 250 mM melatonin. Cultures were stained with the LiverDead Cell-mediated Cytotoxicity Kit Žred color s dead ŽA,C.; green color s live ŽB,D.., viewed on a fluorescence microscope and immediately photographed. All red and green fluorescent cells appear white after photomicrography. Note the abundance of dead cells in control cultures ŽA. and live cells in melatonin-treated cultures ŽD..
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Fig. 2. Photomicrograph of cultures stained with the DNA dye Hoechst 33,258. Control cultures ŽA. contained clumped and fragmented DNA Žopen arrow. associated with apoptotic cells while melatonin treated cultures ŽB. contained nuclear staining typical of healthy cells Žcurved arrow. and few dying cells.
necessary for their survival are provided by the neurons themselves Žautocrine. or by other cells present Žparacrine. in the culture. However, in sparsely seeded cultures, neurons are apparently deprived of sufficient amounts of these essential trophic factors to sustain life. Under these conditions, cells become unhealthy and die via apoptosis within days of growth factor deprivation; mimicking developmental death w27x. To test whether DA neurons similarly undergo developmental cell death when deprived of trophic factors, cells from the E15 rat ventral mesencephalon were seeded at two different densities; high Ž12 500 cellsrmm2 . and low Ž500 cellsrmm2 . and maintained on a chemically defined Žserumrgrowth factor-free. media ŽDM.. As expected, at high density, cells thrived even without the addition of exogenous growth factors. In contrast, cells plated at low density died after only 2–3 days in vitro ŽFig. 1A,B.. By adding substances, such as melatonin, back into the media, it is possible to assess their capacity for salvation. Indeed, when low density cultures were transferred shortly after plating Ž1–2 h. from DM into media supplemented with 250 mM melatonin, little cell death was observed 3–4 days later ŽFig. 1C,D.. That melatonin was
protective for the apoptotic death of growth factor deprived cells was indicated by staining with the DNA dye Hoechst 33,258. Control cultures ŽFig. 2A. contained clumped and fragmented DNA Žopen arrow. associated with apoptotic cells while melatonin treated cultures ŽFig. 2B. contained nuclear staining typical of healthy cells Žcurved arrow. and few dying cells. Since, in the developmental model, all cultured midbrain cells are deprived of necessary growth factors and consequently die, we wondered whether melatonin, using some general mechanism, was rescuing all cells in the dish or was specifically beneficial to certain populations of cells such as the DA neurons. Examination of the specific cell types present in melatonin-rescued cultures revealed the existence of few, if any, glia as assessed by GFAP staining Ždata not shown.. Instead, nearly all majority of cells rescued by melatonin from impending death were neurons containing the neuronal specific marker Tauq ŽFig. 3.. The survival of the specific subset of DA neurons in these cultures was next assessed by immunostaining with antibodies to the DA biosynthetic enzyme TH. We first
Fig. 3. Immunocytochemical localization of Tau in cells from the ventral mesencephalon plated at low density Ž500 cellsrmm2 . and maintained 3–4 days in culture on DM supplemented with 250 mM melatonin. Bar s 50 mm.
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Table 1 Effects of melatonin treatment on the survival of DA and non-DA neurons in midbrain cultures Days in vitro
Melatonin treatment
Total neurons ŽTauq cells.
Percentage survival of total neurons
DA neurons ŽTHq cells.
Percent Survival of DA neurons
0 4 4
y y q
63 428 " 3 802 30 43 765 " 3 177
100 -1 69
3 706 " 194 16 3 761 q 58
100 -1 102
a
Cultures were established as described in the text and stained either for Tau or TH. The number of total neurons ŽTauq cells. and DA neurons ŽTHq cells. were counted in all microscopic fields Ž10 = . 1–2 h after plating or 4 days after growth in DM or 250 mM melatonin.
counted all of the neurons ŽTauq cells. as well as the specific subset of DA neurons ŽTHq cells. present several hours after plating in culture Žcontrol.. At this point, cultures were then split into two groups; untreated ŽDM. and treated Ž250 mM melatonin.. Four days later, cultures were fixed and Tauq and THq cells counted. As detailed in Table 1, 69% of total neurons ŽTauq. survived after melatonin treatment compared to - 1% survival in untreated cultures. Since only 6% of cells present in the culture were THq, most of the neurons saved by melatonin were other than DA neurons. When the survival specifically of DA neurons was examined, however, we unex-
pectedly found that melatonin treatment was more beneficial to DA neurons since all THq neurons were rescued from imminent cell death in these growth factor deprived cultures ŽTable 1: 102% compared to - 1% in untreated cultures; Fig. 4B compared to Fig. 4A.. Not only did DA neurons survive in melatonin but they flourished; eliciting an elaborate network of processes ŽFig. 4C. bearing abundant growth cones ŽFig. 4D.. Parenthetically, in healthy high density cultures, where no added survival benefit was provided by melatonin treatment Ž6924 " 460 THq neurons in DM vs. 6002 " 398 THq neurons in 500 mM melatonin., a more robust neuritic network was observed
Fig. 4. Immunocytochemical localization of TH in ventral mesencephalon neurons plated at low density cultures and fed DM ŽA. or DM supplementaed with 250 mM melatonin ŽB.. Bars in A and B s 50 mm. After 3 days in vitro, cultures were fixed and TH was immunocytochemically localized. Note the striking increase in the number of surviving DA neurons in melatonin-treated cultures as well as the elaborate network of processes ŽC. and growth cones ŽD.. Bars in C and D s 20 mm.
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after melatonin treatment Ždata not shown.. These results suggest that, in addition to its neuroprotective properties, the hormone may also possess neurite-promoting andror reparative effects. We next generated dose-response curves in order to determine the optimal concentrations of melatonin required for the survival of DA neurons in low density cultures. Cells were incubated in media containing increasing concentrations of melatonin, ranging from 1 mM to 500 mM. Four days later, cultures were fixed, TH immunostained and the number of surviving THq neurons counted. A dose-dependent rescue of DA neurons was observed at 4 days in culture with half maximal survival achieved at 8 mM melatonin. No added benefit was derived from incubation at concentrations greater than 250 mM melatonin ŽFig. 5.. In order to examine the time course of these effects, cultures were plated in low density and maintained on media containing 250 mM melatonin Žrefed 2 = srwk.. Cultures were fixed at various time intervals Ž1,3,5,7 days. during treatment and the number of surviving DA neurons tallied. As shown in Fig. 6, THq neurons thrived on melatonin for up to 7 days in vitro, suggesting that the benefits of the hormone were long lasting. We next tested whether continual treatment with melatonin was required for the survival effect. To do so, cells were fed melatonin only during the initial 1 day period in culture, after which it was replaced with DM. The vitality of the cells was monitored daily in the microscope. We found that the cells did not thrive 48 h beyond withdrawal of melatonin ŽFig. 6.. If melatonin is acting as an antioxidant to prevent cell death, then other antioxidants should mimic its effects. To
Fig. 6. Time course of survival in ventral mesencephalon neurons plated in low density and maintained on media containing 250 mM melatonin Žrefed 2=srwk.. Cultures were fixed at various time intervals Ž3,5,7 days. during treatment and the number of surviving DA neurons evaluated immunocytochemically Žsquare.. In other cultures, supplementation with melatonin occurred only during the initial 1 Žcircle. day in culture before replacement with DM. The vitality of the cells was monitored daily in the microscope. Values are expressed as the percentage of TH positive neurons in the culture. Each value represents the mean"S.E.M. of 4 determinations for 2 separate platings. ) P - 0.001.
test this possibility, cultures were established in media containing a variety of antioxidants including, melatonin, the melatonin derivatives, 2-iodomelatonin and 2-iodo-Nbutanoyl-5-methoxytryptamine and the antioxidant vitamins C and E ŽTrolox.. Cultures were fixed 4 days later and the number of DA neurons ŽTHq cells. counted and their process elaboration assessed ŽTable 2.. With the exception of vitamin C, all other compounds were able to rescue similar numbers of THq cells from cell death. However, only melatonin, 2-iodomelatonin and vitamin E treatment resulted in the healthy appearance of neuronal perikarya and the elaboration of long branched varicose processes. In 2-iodo-N-butanoyl-5-methoxytryptamine treated cultures, although a similar number of THq cells were present, most were small and unhealthy looking. Vitamin C was only partially able to block cell death. Like 2-iodo-N-butanoyl-5-methoxytryptamine, few of the remaining cells in vitamin C treated cultures were processbearing. 3.2. Effects of melatonin on the MPP q model
Fig. 5. Dose-dependency of the survival effect of melatonin on E15 cultured ventral mesencephalon neurons plated int low density cultures. One hour after plating, cultures were rinsed and fed media supplemented with increasing concentrations of melatonin, ranging from 1 to 500 mM. Four days later, cultures were fixed, TH immunostained and the number of surviving THq neurons counted. Values are expressed as the percentage of TH positive neurons in the culture. Each value represents the mean"S.E.M. of 4 determinations for 2 separate platings.
The next series of experiments were designed to assess the potential for melatonin to rescue DA neurons from the oxidative injuryrdeath caused by the DA-specific neurotoxin MPPq. Dissociated cells from E15 rat ventral mesencephalon were plated in culture at high density Ž12 500 cellsrmm2 .. Under these conditions, survival can be maintained indefinitely on DM. On day 3, cultures were transferred into media containing 2.5 mM MPPq. Twenty-four hours later, MPPq was removed and replaced with media
L. IacoÕitti et al.r Brain Research 768 (1997) 317–326 Table 2 Effects of a variety of antioxidants on the survival of DA neurons in growth factor-deprived cultures Treatment
Concentration ŽmM.
a THq neurons
Process formation
Melatonin
250 100 10 250 100 10 250 100 10 250 100 10 250 100 10
3 084"280 2 872"212 2 274"196 2 612"228 3 332"269 3 076"188 3 382"292 3 007"167 3 257"314 3 196"201 3 064"278 3 051q199 67" 45 343" 68 879q121
qqqq qqqq qqq qqq qqq qq qqqq qqqq qqq y q q y q q
2-Iodomelatonin
Vitamin E
2-IbMT
Vitamin C
Cultures were replenished daily with media containing 10–250 mM melatonin, 2-iodomelatonin, 2-iodo-N-butanoyl-5-methoxytryptamine Ž2IbMT., vitamin C or the water soluble vitamin E analog, Trolox. Cultures were fixed 4 days later and the number of DA neurons ŽTHq cells. counted in all 10= microscopic fields. In addition, since process formation varied from culture to culture, each was assessed using a rating scale where y indicates no processes; q to qqqq indicates increasing neuritic arborization.
containing various concentrations of melatonin Ž10 mM to 500 mM.. Cultures were fixed and processed for TH immunocytochemistry three days later. As found previ-
Fig. 7. Dose-dependency of the survival effect of melatonin on E15 cultured ventral mesencephalon neurons damaged by MPPq exposure. Dissociated cells were plated in culture at high density Ž12 500 cellsrmm2 . and maintained on DM until day 3 when they were transferred into DM containing 2.5 mM MPPq. Twenty-four hours later, MPPq was removed and replaced with DM containing various concentrations of melatonin Ž10–500 mM.. Three days later, cultures were fixed, TH immunostained and the number of surviving THq neurons counted. As indicated, MPPq produced a 50% loss of THq neurons. Values are expressed as the percentage of THq neurons present in control Žno MPPq . cultures. Each value represents the mean"S.E.M. of 4 determinations for 2 separate platings. ) P - 0.001.
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ously using this experimental paradigm, MPPq resulted in a reproducible 50% loss of DA neurons 3 days after treatment w43x. Supplementation of toxin-treated cultures with melatonin resulted in the dose-dependent salvation of DA neurons ŽFig. 7.. In cultures receiving maximal concentrations Ž500 mM., near total recovery of THq cell numbers was observed. These results suggest that the oxidative stress placed on DA neurons by MPPq may be counteracted by subsequent treatment with the antioxidant melatonin.
4. Discussion The present study demonstrates that the potent antioxidant hormone melatonin possesses an unprecedented ability to rescue midbrain neurons from the apoptotic death which follows the withdrawal of essential growth factors from their culture media or treatment with the neurotoxin MPPq. While cell viability can be sustained for extended periods in culture by continual growth in melatonin-containing media, this beneficial effect is both dose and time dependent. The mechanism by which melatonin achieves these remarkable effects has not been studied here. While the prospect that melatonin mediates its effects through classic membrane receptors wfor review; w3,32,44,50xx is still under investigation, the more plausable alternative is that, following diffusion into the cell, melatonin rescues neurons from death by defending them against damaging oxyradicals. Supplying proof of this antioxidant action is, however, particularly difficult in our system, since, regardless of its mechanism of action Žantioxidant or otherwise., oxyradicals would be expected to be present in lower concentration in melatonin-treated cultures where cells are healthy than in control Žgrowth factor-deprived or toxin-treated. cultures where cells are dying. Nonetheless, several lines of evidence provide circumstantial support for this notion, including: Ža. concentrations of melatonin much greater than those required for melatonin receptor binding are needed for the rescue of DA neurons in culture; Žb. 2-ibMT, which is a poor melatonin receptor agonist, is also protective of DA neurons; and Žc. other classes of antioxidants, like vitamin E, similarly rescue dying DA neurons. Further suggestive of an antioxidant role for melatonin is the fact that the cell damagerdeath observed in both experimental models has been associated with oxidative crisis. This proposition is consistent with the finding that, in the developmental model, cells ŽDA neurons, other neurons and glia. maintained in culture under sub-optimal conditions Ži.e. low density, no serumrgrowth factors. require melatonin for their survival while those grown with minimal stress Žat high density or in the presence of serumrgrowth factors. do not. Presumably, by neutralizing the build-up of oxyradicals that accompanies growth factor
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deprivation, melatonin interrupts the suicide program that causes apoptotic death in such beleaguered neurons. In a similar fashion, the apoptosis that peripheral neurons undergo following nerve growth factor withdrawal can be blocked by over-expression of SOD w15x or the bcl-2 gene product w13,16x, both of which are antioxidants w6,22,22,27,39,40x. Peripheral neurons w16x, however, outgrow their dependence on growth factors and consequently their requirement for intervention by antioxidants. In contrast, DA neurons require continued melatonin to sustain life, indicating that, even at later times in culture, apoptosis remains a necessary fate of DA neurons deprived of essential growth factors. Likewise, melatonin’s ability to salvage MPPq-treated DA neurons likely stems from its capacity to de-toxify oxyradicals generated by neurotoxin treatment. Supporting this notion is the recent discovery that transgenic mice with increased levels of CurZn SOD activity are resistant to the toxicity of MPPq w37x. That antioxidants can play a useful role in the protection of nigral DA neurons from neurotoxic injury has been illustrated previously with antioxidative agents, such as, ascorbate, deprenyl, vitamin E, and DMSO w5,8,51x. In the MPPq experiments, where the neurotoxin specifically kills DA neurons, there is no question of the specificity of melatonin’s effects. However, in the developmental model where all cultured midbrain cells are adversely impacted, melatonin saves both DA and non-DA neurons. Contrary to our expectations, however, treatment is more beneficial in the former class of cells than in the latter Ž102% of DA neurons compared to 69% survival of total neurons.. One would have anticipated that the proportion of rescued DA neurons would decline not increase since the number of Tauq ŽTHy. cells in the E15 midbrain should rise as many of these cells continue to divide during the 4 day culture period while the number of THq neurons, because they are postmitotic w41x, remains unchanged. It has been hypothesized that DA neurons are more vulnerable to oxidative damage than other neurons wfor review; w12xx. Possibly, this vulnerability also makes them more amenable to salvation by antioxidants. Alternatively, it may suggest that melatonin is working through a distinct mechanism of action in DA neurons. In addition to the present study on DA and non-DA midbrain neurons, several previous studies have described neuroprotection of other damaged brain neurons by melatonin. Thus, in one study, melatonin was shown to protect cerebellar or cortical neurons from kainate-induced excitotoxicity in vitro w17x and in vivo w18x. The mechanism of this neuroprotective action was not determined, but apparently did not require a direct effect of melatonin on glutamate receptors. In a correlative study, melatonin also protected cerebellar neurons from the apoptotic death that results when oxygen free radicals are generated after treatment with the photosensitive dye, rose bengal w29x. Since oxidative stress accompanies glutamate excitotoxicity and
dye-induced apoptosis, the antioxidant action of melatonin was thought to play an important role in the improved neuronal survival observed in these studies. Despite the hormone’s enormous lay popularity, there is a dearth of information on melatonin’s antioxidant actions in the nervous system. Even less is known about melatonin’s other potential roles Ži.e. growth factor-like effects. which may ultimately prove to be important in the rescue of cells from damagerdeath. The present investigation represents the first attempt to study melatonin’s potential utility in protecting specifically DA neurons from cell death. The hope is that the discovery of substances capable of rescuing cultured neurons from developmental or neurotoxic death will, by extrapolation, be useful in halting the progressive loss of neurons during aging or after injury or disease. At present, a variety of endogenous substances, including gangliosides, growth factors, vitamins etc., are being considered as pharmacological agents for the treatment of Parkinson’s Disease. Despite the fact that these substances may already be present at physiological levels in the diseased brain, their exogenous application has, in some instances, proven beneficial to injured DA neurons both in vitro and in vivo w2,34,43,47,48x. Nonetheless, there remains no known way to prevent the loss of DA neurons in diseases like Parkinson’s. Whether melatonin’s usefulness in protecting DA neurons in vitro translates into an in vivo role for the molecule in the treatment of Parkinson’s Disease awaits further investigation.
Acknowledgements This work was supported by NIH NS32519 to L.I.
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Research report
Melatonin rescues dopamine neurons from cell death in tissue culture models of oxidative stress Lorraine Iacovitti ) , Natalie D. Stull, Kelly Johnston Department of Neurobiology and Anatomy, Mailstop 408, Allegheny UniÕersity of the Health Sciences, Broad and Vine Streets, Philadelphia, PA 19102, USA Accepted 13 May 1997
Abstract Dopamine ŽDA. neurons are uniquely vulnerable to damage and disease. Their loss in humans is associated with diseases of the aged, most notably, Parkinson’s Disease ŽPD.. There is now a great deal of evidence to suggest that the destruction of DA neurons in PD involves the accumulation of harmful oxygen free radicals. Since the antioxidant hormone, melatonin, is one of the most potent endogenous scavengers of these toxic radicals, we tested its ability to rescue DA neurons from damagerdeath in several laboratory models associated with oxidative stress. In the first model, cells were grown in low density on serum-free media. Under these conditions, nearly all cells died, presumably due to the lack of essential growth factors. Treatment with 250 mM melatonin rescued nearly all dying cells Ž100% tauq neurons., including tyrosine hydroxylase immunopositive DA neurons, for at least 7 days following growth factor deprivation. This effect was dose and time dependent and was mimicked by other antioxidants such as 2-iodomelatonin and vitamin E. Similarly, in the second model of oxidative stress, 250 mM melatonn produced a near total recovery from the usual 50% loss of DA neurons caused by neurotoxic injury from 2.5 mM 1-methyl-4-phenylpyridine ŽMPPq.. These results indicate that melatonin possesses the remarkable ability to rescue DA neurons from cell death in several experimental paradigms associated with oxidative stress. q 1997 Elsevier Science B.V. Keywords: Melatonin; Oxidative stress; Dopamine neuron; Tissue culture; MPPq; Oxyradical
1. Introduction Almost a century has passed since it was first recognized that Parkinson’s Disease ŽPD. involved the selective loss of neurons from specific nuclei in the ventral midbrain region w49x. Some forty years later, Ehringer and Hornykiewicz Ž1960. w11x demonstrated that the affected cells used dopamine ŽDA. as their neurotransmitter. The most severe loss of DA neurons occurs in the substantia nigra ŽSN.; with more moderate losses in the ventral tegmental area and A8 neurons w20x. Although a number of different theories have been advanced over the years to explain the selective vulnerability of DA neurons in PD, the mechanism of cell destruction and the cause of the disease remain a mystery. One intriguing theory holds that there is cytotoxic loss of DA neurons following oxidative stress to the cells caused by the formation of hydrogen peroxide ŽH 2 O 2 . and ) Corresponding author. Fax: q1 Ž215. 762-3127; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 6 8 - 9
oxygen free radicals Žfor review; w12x.. Indeed, there is an impressive body of evidence to suggest that DA neurons of the SN are at greater risk for oxidative stress and more susceptible to its ravages than other structures in the brain. Firstly, neurons of the SN contain neuromelanin, an autooxidative byproduct of DA metabolism w14x which can lead to the formation of superoxides. Under normal conditions, these oxyradicals are de-toxified by cytosolic CurZn superoxide dismutase ŽSOD.. The fact that SOD mRNA is present in higher than normal levels in PD is suggestive of an increased requirement for superoxide scavengers in diseased DA neurons w4x. Moreover, since SOD itself leads to the production of H 2 O 2 , its increased activity in PD is likely to further exacerbate the stress placed on these cells w35x. Contributing further to their risk of oxidative crisis, is the fact that the normal antioxidative defense system Žcatalase and glutathione peroxidase. used to de-toxify H 2 O 2 is reduced in PD w12x and in the aged brain w7x; resulting in an accumulation of highly toxic hydroxyl radicals. Likewise, iron, which also participates in the formation of hydroxyl radicals from H 2 O 2 , is increased in
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DA neurons in PD w8,10x. These toxic species, in turn, cause lipid peroxidation, provoking disturbances in cell membranes, calcium influx, disruption of the cytoskeleton and a cascade of events leading to cell death w20x. Experimental paradigms mimicking these events in culture make possible the search for substances of potential importance in neuronal salvation from oxidative damage andror death. The first of these models is based upon the belief that, during development, neurons compete for trophic substances found in target tissues. Because these factors are available in limited supply, many neurons are unsuccessful in their bid and consequently initiate a suicide program resulting in their demise. Developmental cell death can be modeled in vitro by depriving cells in culture of these same essential factors. Under these conditions, the general health of cells declines rapidly, resulting in apoptosis Žincluding cell shrinkage, nuclear condensation, DNA fragmentation, blebbing of the plasma membrane. within approximately 48 h wfor review, w26xx. The evidence that oxidative stress underlies developmental apoptosis from growth factor deprivation derives from an elegant series of experiments by Johnson and colleagues. These studies established that the overexpression of SOD w15x or the bcl-2 gene product w13,16x, both of which are antioxidants w6,21,22,27,39,40x, prevents the apoptosis that peripheral neurons undergo following nerve growth factor withdrawal. Although the trigger for cell destruction is quite different from trophic factor deprivation, treatment with the specific CA neurotoxin MPPq w19x represents a second important experimental paradigm for evaluating a substance’s ability to rescue neurons from oxidative cell death. MPPq is thought to exert its toxic effects by impairing complex I of the mitochodrial respiratory chain w42x which induces increased amounts of superoxide radicals, lipid peroxidation w12x homeostatic changes in Ca2q w28,38x and, finally, apoptosis w31x. It is not surprising, in light of the important role that oxygen free radicals play in producing these toxic effects Žfor review, Fahn and Cohen, 1992 w12x., that the use of antioxidants has become an important new line of investigation. Indeed, several antioxidants Žvitamins A and E. can, with varying degrees of success, protect nigral DA neurons from neurotoxin-induced damage w5,51x. Arguably, the most potent and versatile of cellular antioxidants is the pineal hormone, melatonin Ž5-methoxyN-acetyl-tryptamine., which possesses multiple ways to both reduce free radical production and to neutralize radicals once generated w34x. These effects are accomplished without receptors w30x, presumably because of the indole’s high lipid solubility which allows it free access to the interior of the cell. Typical of a hormone, and in contrast to vitamin antioxidants which are compartmentalized, melatonin is capable of acting in the membrane, cytosol and nucleus w30x. Studies in vitro have demonstrated a remarkable capacity for melatonin to scavenge hydroxyl
w45,46x and peroxyl radicals w33x. Furthermore, melatonin stimulates the antioxidant enzyme, glutathione peroxidase w1x while inhibiting nitric oxide synthase from generating more free radicals w36x. Although virtually nothing is known regarding melatonin’s effects on the DA system, its extraordinary antioxidant properties suggested to us a potential role for melatonin in the rescue of DA neurons damaged by oxidative stress. We will show that melatonin treatment can dramatically salvage DA neurons from the inevitable cell death caused by oxidative stress due to growth factor deprivation or MPPq treatment. 2. Materials and methods 2.1. Tissue culture Pregnant Sprague-Dawley rats were purchased from Taconic Laboratories at 15 days gestation q12 h Žfertilization day s embryonic day 0.. Pregnant dams were deeply anaesthetized with 40 mgrkg sodium pentobarbital while embryos were removed and transferred into Leibowitz-15 medium ŽL-15. for dissection. The developing ventral mesencephalon Ž1 mm2 . was isolated from the remainder of the brain as described previously w25x. After removal of the meninges, tissue was minced and incubated in Ca2q-, Mg 2q-free Hank’s balanced salt solution ŽCMF-HBSS. for 8 min at 378C in a clinical rotator Ž40 rpm.. The incubation mixture was replaced with a 0.01% trypsin solution Žin CMF-HBSS., and incubated for an additional 8 min, rinsed twice in Leibovitz medium ŽL-15., and placed in culture medium containing Dulbecco’s minimum essential medium ŽDMEM., 10% fetal calf serum, glucose Ž6 mgrml., glutamine Ž204 mgrml., and penicillinrstreptomycin Ž100 Urml.. Cells were dissociated by trituration through a reduced bore glass pipette and plated onto plastic Lab Tek 8-well culture slides coated with polymerized polyornithine Žprecoated overnight at 378C at a concentration of 0.01 mgrml in 15 mM borate buffer, pH 8.4. then rinsed with water and air-dried 5 min. The cells were plated at two different densities; high Ž12500 cellsrmm2 . and low Ž500 cellsrmm2 .. After a 1–2 h stabilization period in standard media, cultures were transferred to a chemically defined serum-free media ŽDM. containing 50% DMEM, 50% Ham’s F12 media, 1% ITSq, and glucose, glutamine and penicillinrstreptomycin as described above. 2.2. Cell Õiability and cytotoxicity assays Cell viability and cytotoxicity were assessed based on the principle that live cells contain ubiquitous intracellular esterase activity which can be detected by the enzymatic conversion of the nonfluorescent cell permeant calcein AM to the highly fluorescent calcein Žgreen fluorescence at 530 nm.. Dead cells were detected after ethydium homodimer entered cells through damaged membranes to bind to
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DNA, making them fluoresce red Ž) 600 nm.. Essentially cultured cells were rinsed in PBS, 100 ml of combined LIVErDEAD assay reagents Žfinal concentration 0.5 mM calcein AM and ethydium homodimer. were added and incubated for 45 min at room temperature. Cultures were then viewed on a fluorescence microscope. Apoptosis was assessed using the DNA dye Hoescht 33,258; unhealthy apoptotic cells contain clumped and fragmented DNA while healthy cells do not. 2.3. Immunocytochemistry Cultures were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4. and processed with antibodies to TH Ž1 : 5000 dilution., glial fibrillary acidic protein ŽGFAP. Ž1 : 400 made in mouse. or Tau Ž1 : 200 made in rabbit. using the immunoperoxidaserABC method of staining ŽElite Vectakit. as adapted for tissue culture w24x. The number of immunoreactive neurons was determined by counting positively stained cells in all microscopic fields on the culture dish. This was accomplished with the aid of an eyepiece reticule used at a 10 = magnification.
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ferences were considered significant only when the P value was less than 0.05. 2.5. Chemical reagents GFAP and Tau antibodies were purchased from Sigma Chemical ŽSt. Louis, MO, USA.. TH antibodies were a kind gift of Dr T.H. Joh, New York, NY, USA. All reagents used in culture were obtained from the Gibco Co. or Fisher Scientific with the exception of ITSq purchased from Collaborative Biomedical Products and fetal calf serum from Irvine Scientific. Melatonin ŽCat. a 5250. was purchased from Sigma Chemical ŽSt. Louis, MO, USA.; 2-iodomelatonin ŽCat.a 550-153-M005. from Alexis Corp.; 2-iodo-N-butanoyl-5-methoxytryptamine from RBI ŽCat. a M-112. and the water soluble vitamin E analog, Trolox Ž6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid. from Aldrich Co. Liverdead Cell-mediated Cytotoxicity Kit was purchased from Molecular Probes Inc. ŽEugene, OR, USA..
3. Results 2.4. Statistical analysis 3.1. Effects of melatonin on the deÕelopmental model Data were statistically analyzed by one way analysis of variance. When P - 0.05, then the F-test was followed by the two-tailed Student’s t-test to compare the statistical significance between control and experimental groups. Dif-
In general, neurons flourish in culture even in the absence of exogenous growth factors if they are plated at a sufficiently high density. Presumably the trophic factors
Fig. 1. Photomicrograph of cells from the E15 rat ventral mesencephalon plated at low density Ž500 cellsrmm2 . and maintained for 3–4 days in vitro on DM ŽA,B. or DM supplemented with 250 mM melatonin. Cultures were stained with the LiverDead Cell-mediated Cytotoxicity Kit Žred color s dead ŽA,C.; green color s live ŽB,D.., viewed on a fluorescence microscope and immediately photographed. All red and green fluorescent cells appear white after photomicrography. Note the abundance of dead cells in control cultures ŽA. and live cells in melatonin-treated cultures ŽD..
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Fig. 2. Photomicrograph of cultures stained with the DNA dye Hoechst 33,258. Control cultures ŽA. contained clumped and fragmented DNA Žopen arrow. associated with apoptotic cells while melatonin treated cultures ŽB. contained nuclear staining typical of healthy cells Žcurved arrow. and few dying cells.
necessary for their survival are provided by the neurons themselves Žautocrine. or by other cells present Žparacrine. in the culture. However, in sparsely seeded cultures, neurons are apparently deprived of sufficient amounts of these essential trophic factors to sustain life. Under these conditions, cells become unhealthy and die via apoptosis within days of growth factor deprivation; mimicking developmental death w27x. To test whether DA neurons similarly undergo developmental cell death when deprived of trophic factors, cells from the E15 rat ventral mesencephalon were seeded at two different densities; high Ž12 500 cellsrmm2 . and low Ž500 cellsrmm2 . and maintained on a chemically defined Žserumrgrowth factor-free. media ŽDM.. As expected, at high density, cells thrived even without the addition of exogenous growth factors. In contrast, cells plated at low density died after only 2–3 days in vitro ŽFig. 1A,B.. By adding substances, such as melatonin, back into the media, it is possible to assess their capacity for salvation. Indeed, when low density cultures were transferred shortly after plating Ž1–2 h. from DM into media supplemented with 250 mM melatonin, little cell death was observed 3–4 days later ŽFig. 1C,D.. That melatonin was
protective for the apoptotic death of growth factor deprived cells was indicated by staining with the DNA dye Hoechst 33,258. Control cultures ŽFig. 2A. contained clumped and fragmented DNA Žopen arrow. associated with apoptotic cells while melatonin treated cultures ŽFig. 2B. contained nuclear staining typical of healthy cells Žcurved arrow. and few dying cells. Since, in the developmental model, all cultured midbrain cells are deprived of necessary growth factors and consequently die, we wondered whether melatonin, using some general mechanism, was rescuing all cells in the dish or was specifically beneficial to certain populations of cells such as the DA neurons. Examination of the specific cell types present in melatonin-rescued cultures revealed the existence of few, if any, glia as assessed by GFAP staining Ždata not shown.. Instead, nearly all majority of cells rescued by melatonin from impending death were neurons containing the neuronal specific marker Tauq ŽFig. 3.. The survival of the specific subset of DA neurons in these cultures was next assessed by immunostaining with antibodies to the DA biosynthetic enzyme TH. We first
Fig. 3. Immunocytochemical localization of Tau in cells from the ventral mesencephalon plated at low density Ž500 cellsrmm2 . and maintained 3–4 days in culture on DM supplemented with 250 mM melatonin. Bar s 50 mm.
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Table 1 Effects of melatonin treatment on the survival of DA and non-DA neurons in midbrain cultures Days in vitro
Melatonin treatment
Total neurons ŽTauq cells.
Percentage survival of total neurons
DA neurons ŽTHq cells.
Percent Survival of DA neurons
0 4 4
y y q
63 428 " 3 802 30 43 765 " 3 177
100 -1 69
3 706 " 194 16 3 761 q 58
100 -1 102
a
Cultures were established as described in the text and stained either for Tau or TH. The number of total neurons ŽTauq cells. and DA neurons ŽTHq cells. were counted in all microscopic fields Ž10 = . 1–2 h after plating or 4 days after growth in DM or 250 mM melatonin.
counted all of the neurons ŽTauq cells. as well as the specific subset of DA neurons ŽTHq cells. present several hours after plating in culture Žcontrol.. At this point, cultures were then split into two groups; untreated ŽDM. and treated Ž250 mM melatonin.. Four days later, cultures were fixed and Tauq and THq cells counted. As detailed in Table 1, 69% of total neurons ŽTauq. survived after melatonin treatment compared to - 1% survival in untreated cultures. Since only 6% of cells present in the culture were THq, most of the neurons saved by melatonin were other than DA neurons. When the survival specifically of DA neurons was examined, however, we unex-
pectedly found that melatonin treatment was more beneficial to DA neurons since all THq neurons were rescued from imminent cell death in these growth factor deprived cultures ŽTable 1: 102% compared to - 1% in untreated cultures; Fig. 4B compared to Fig. 4A.. Not only did DA neurons survive in melatonin but they flourished; eliciting an elaborate network of processes ŽFig. 4C. bearing abundant growth cones ŽFig. 4D.. Parenthetically, in healthy high density cultures, where no added survival benefit was provided by melatonin treatment Ž6924 " 460 THq neurons in DM vs. 6002 " 398 THq neurons in 500 mM melatonin., a more robust neuritic network was observed
Fig. 4. Immunocytochemical localization of TH in ventral mesencephalon neurons plated at low density cultures and fed DM ŽA. or DM supplementaed with 250 mM melatonin ŽB.. Bars in A and B s 50 mm. After 3 days in vitro, cultures were fixed and TH was immunocytochemically localized. Note the striking increase in the number of surviving DA neurons in melatonin-treated cultures as well as the elaborate network of processes ŽC. and growth cones ŽD.. Bars in C and D s 20 mm.
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after melatonin treatment Ždata not shown.. These results suggest that, in addition to its neuroprotective properties, the hormone may also possess neurite-promoting andror reparative effects. We next generated dose-response curves in order to determine the optimal concentrations of melatonin required for the survival of DA neurons in low density cultures. Cells were incubated in media containing increasing concentrations of melatonin, ranging from 1 mM to 500 mM. Four days later, cultures were fixed, TH immunostained and the number of surviving THq neurons counted. A dose-dependent rescue of DA neurons was observed at 4 days in culture with half maximal survival achieved at 8 mM melatonin. No added benefit was derived from incubation at concentrations greater than 250 mM melatonin ŽFig. 5.. In order to examine the time course of these effects, cultures were plated in low density and maintained on media containing 250 mM melatonin Žrefed 2 = srwk.. Cultures were fixed at various time intervals Ž1,3,5,7 days. during treatment and the number of surviving DA neurons tallied. As shown in Fig. 6, THq neurons thrived on melatonin for up to 7 days in vitro, suggesting that the benefits of the hormone were long lasting. We next tested whether continual treatment with melatonin was required for the survival effect. To do so, cells were fed melatonin only during the initial 1 day period in culture, after which it was replaced with DM. The vitality of the cells was monitored daily in the microscope. We found that the cells did not thrive 48 h beyond withdrawal of melatonin ŽFig. 6.. If melatonin is acting as an antioxidant to prevent cell death, then other antioxidants should mimic its effects. To
Fig. 6. Time course of survival in ventral mesencephalon neurons plated in low density and maintained on media containing 250 mM melatonin Žrefed 2=srwk.. Cultures were fixed at various time intervals Ž3,5,7 days. during treatment and the number of surviving DA neurons evaluated immunocytochemically Žsquare.. In other cultures, supplementation with melatonin occurred only during the initial 1 Žcircle. day in culture before replacement with DM. The vitality of the cells was monitored daily in the microscope. Values are expressed as the percentage of TH positive neurons in the culture. Each value represents the mean"S.E.M. of 4 determinations for 2 separate platings. ) P - 0.001.
test this possibility, cultures were established in media containing a variety of antioxidants including, melatonin, the melatonin derivatives, 2-iodomelatonin and 2-iodo-Nbutanoyl-5-methoxytryptamine and the antioxidant vitamins C and E ŽTrolox.. Cultures were fixed 4 days later and the number of DA neurons ŽTHq cells. counted and their process elaboration assessed ŽTable 2.. With the exception of vitamin C, all other compounds were able to rescue similar numbers of THq cells from cell death. However, only melatonin, 2-iodomelatonin and vitamin E treatment resulted in the healthy appearance of neuronal perikarya and the elaboration of long branched varicose processes. In 2-iodo-N-butanoyl-5-methoxytryptamine treated cultures, although a similar number of THq cells were present, most were small and unhealthy looking. Vitamin C was only partially able to block cell death. Like 2-iodo-N-butanoyl-5-methoxytryptamine, few of the remaining cells in vitamin C treated cultures were processbearing. 3.2. Effects of melatonin on the MPP q model
Fig. 5. Dose-dependency of the survival effect of melatonin on E15 cultured ventral mesencephalon neurons plated int low density cultures. One hour after plating, cultures were rinsed and fed media supplemented with increasing concentrations of melatonin, ranging from 1 to 500 mM. Four days later, cultures were fixed, TH immunostained and the number of surviving THq neurons counted. Values are expressed as the percentage of TH positive neurons in the culture. Each value represents the mean"S.E.M. of 4 determinations for 2 separate platings.
The next series of experiments were designed to assess the potential for melatonin to rescue DA neurons from the oxidative injuryrdeath caused by the DA-specific neurotoxin MPPq. Dissociated cells from E15 rat ventral mesencephalon were plated in culture at high density Ž12 500 cellsrmm2 .. Under these conditions, survival can be maintained indefinitely on DM. On day 3, cultures were transferred into media containing 2.5 mM MPPq. Twenty-four hours later, MPPq was removed and replaced with media
L. IacoÕitti et al.r Brain Research 768 (1997) 317–326 Table 2 Effects of a variety of antioxidants on the survival of DA neurons in growth factor-deprived cultures Treatment
Concentration ŽmM.
a THq neurons
Process formation
Melatonin
250 100 10 250 100 10 250 100 10 250 100 10 250 100 10
3 084"280 2 872"212 2 274"196 2 612"228 3 332"269 3 076"188 3 382"292 3 007"167 3 257"314 3 196"201 3 064"278 3 051q199 67" 45 343" 68 879q121
qqqq qqqq qqq qqq qqq qq qqqq qqqq qqq y q q y q q
2-Iodomelatonin
Vitamin E
2-IbMT
Vitamin C
Cultures were replenished daily with media containing 10–250 mM melatonin, 2-iodomelatonin, 2-iodo-N-butanoyl-5-methoxytryptamine Ž2IbMT., vitamin C or the water soluble vitamin E analog, Trolox. Cultures were fixed 4 days later and the number of DA neurons ŽTHq cells. counted in all 10= microscopic fields. In addition, since process formation varied from culture to culture, each was assessed using a rating scale where y indicates no processes; q to qqqq indicates increasing neuritic arborization.
containing various concentrations of melatonin Ž10 mM to 500 mM.. Cultures were fixed and processed for TH immunocytochemistry three days later. As found previ-
Fig. 7. Dose-dependency of the survival effect of melatonin on E15 cultured ventral mesencephalon neurons damaged by MPPq exposure. Dissociated cells were plated in culture at high density Ž12 500 cellsrmm2 . and maintained on DM until day 3 when they were transferred into DM containing 2.5 mM MPPq. Twenty-four hours later, MPPq was removed and replaced with DM containing various concentrations of melatonin Ž10–500 mM.. Three days later, cultures were fixed, TH immunostained and the number of surviving THq neurons counted. As indicated, MPPq produced a 50% loss of THq neurons. Values are expressed as the percentage of THq neurons present in control Žno MPPq . cultures. Each value represents the mean"S.E.M. of 4 determinations for 2 separate platings. ) P - 0.001.
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ously using this experimental paradigm, MPPq resulted in a reproducible 50% loss of DA neurons 3 days after treatment w43x. Supplementation of toxin-treated cultures with melatonin resulted in the dose-dependent salvation of DA neurons ŽFig. 7.. In cultures receiving maximal concentrations Ž500 mM., near total recovery of THq cell numbers was observed. These results suggest that the oxidative stress placed on DA neurons by MPPq may be counteracted by subsequent treatment with the antioxidant melatonin.
4. Discussion The present study demonstrates that the potent antioxidant hormone melatonin possesses an unprecedented ability to rescue midbrain neurons from the apoptotic death which follows the withdrawal of essential growth factors from their culture media or treatment with the neurotoxin MPPq. While cell viability can be sustained for extended periods in culture by continual growth in melatonin-containing media, this beneficial effect is both dose and time dependent. The mechanism by which melatonin achieves these remarkable effects has not been studied here. While the prospect that melatonin mediates its effects through classic membrane receptors wfor review; w3,32,44,50xx is still under investigation, the more plausable alternative is that, following diffusion into the cell, melatonin rescues neurons from death by defending them against damaging oxyradicals. Supplying proof of this antioxidant action is, however, particularly difficult in our system, since, regardless of its mechanism of action Žantioxidant or otherwise., oxyradicals would be expected to be present in lower concentration in melatonin-treated cultures where cells are healthy than in control Žgrowth factor-deprived or toxin-treated. cultures where cells are dying. Nonetheless, several lines of evidence provide circumstantial support for this notion, including: Ža. concentrations of melatonin much greater than those required for melatonin receptor binding are needed for the rescue of DA neurons in culture; Žb. 2-ibMT, which is a poor melatonin receptor agonist, is also protective of DA neurons; and Žc. other classes of antioxidants, like vitamin E, similarly rescue dying DA neurons. Further suggestive of an antioxidant role for melatonin is the fact that the cell damagerdeath observed in both experimental models has been associated with oxidative crisis. This proposition is consistent with the finding that, in the developmental model, cells ŽDA neurons, other neurons and glia. maintained in culture under sub-optimal conditions Ži.e. low density, no serumrgrowth factors. require melatonin for their survival while those grown with minimal stress Žat high density or in the presence of serumrgrowth factors. do not. Presumably, by neutralizing the build-up of oxyradicals that accompanies growth factor
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deprivation, melatonin interrupts the suicide program that causes apoptotic death in such beleaguered neurons. In a similar fashion, the apoptosis that peripheral neurons undergo following nerve growth factor withdrawal can be blocked by over-expression of SOD w15x or the bcl-2 gene product w13,16x, both of which are antioxidants w6,22,22,27,39,40x. Peripheral neurons w16x, however, outgrow their dependence on growth factors and consequently their requirement for intervention by antioxidants. In contrast, DA neurons require continued melatonin to sustain life, indicating that, even at later times in culture, apoptosis remains a necessary fate of DA neurons deprived of essential growth factors. Likewise, melatonin’s ability to salvage MPPq-treated DA neurons likely stems from its capacity to de-toxify oxyradicals generated by neurotoxin treatment. Supporting this notion is the recent discovery that transgenic mice with increased levels of CurZn SOD activity are resistant to the toxicity of MPPq w37x. That antioxidants can play a useful role in the protection of nigral DA neurons from neurotoxic injury has been illustrated previously with antioxidative agents, such as, ascorbate, deprenyl, vitamin E, and DMSO w5,8,51x. In the MPPq experiments, where the neurotoxin specifically kills DA neurons, there is no question of the specificity of melatonin’s effects. However, in the developmental model where all cultured midbrain cells are adversely impacted, melatonin saves both DA and non-DA neurons. Contrary to our expectations, however, treatment is more beneficial in the former class of cells than in the latter Ž102% of DA neurons compared to 69% survival of total neurons.. One would have anticipated that the proportion of rescued DA neurons would decline not increase since the number of Tauq ŽTHy. cells in the E15 midbrain should rise as many of these cells continue to divide during the 4 day culture period while the number of THq neurons, because they are postmitotic w41x, remains unchanged. It has been hypothesized that DA neurons are more vulnerable to oxidative damage than other neurons wfor review; w12xx. Possibly, this vulnerability also makes them more amenable to salvation by antioxidants. Alternatively, it may suggest that melatonin is working through a distinct mechanism of action in DA neurons. In addition to the present study on DA and non-DA midbrain neurons, several previous studies have described neuroprotection of other damaged brain neurons by melatonin. Thus, in one study, melatonin was shown to protect cerebellar or cortical neurons from kainate-induced excitotoxicity in vitro w17x and in vivo w18x. The mechanism of this neuroprotective action was not determined, but apparently did not require a direct effect of melatonin on glutamate receptors. In a correlative study, melatonin also protected cerebellar neurons from the apoptotic death that results when oxygen free radicals are generated after treatment with the photosensitive dye, rose bengal w29x. Since oxidative stress accompanies glutamate excitotoxicity and
dye-induced apoptosis, the antioxidant action of melatonin was thought to play an important role in the improved neuronal survival observed in these studies. Despite the hormone’s enormous lay popularity, there is a dearth of information on melatonin’s antioxidant actions in the nervous system. Even less is known about melatonin’s other potential roles Ži.e. growth factor-like effects. which may ultimately prove to be important in the rescue of cells from damagerdeath. The present investigation represents the first attempt to study melatonin’s potential utility in protecting specifically DA neurons from cell death. The hope is that the discovery of substances capable of rescuing cultured neurons from developmental or neurotoxic death will, by extrapolation, be useful in halting the progressive loss of neurons during aging or after injury or disease. At present, a variety of endogenous substances, including gangliosides, growth factors, vitamins etc., are being considered as pharmacological agents for the treatment of Parkinson’s Disease. Despite the fact that these substances may already be present at physiological levels in the diseased brain, their exogenous application has, in some instances, proven beneficial to injured DA neurons both in vitro and in vivo w2,34,43,47,48x. Nonetheless, there remains no known way to prevent the loss of DA neurons in diseases like Parkinson’s. Whether melatonin’s usefulness in protecting DA neurons in vitro translates into an in vivo role for the molecule in the treatment of Parkinson’s Disease awaits further investigation.
Acknowledgements This work was supported by NIH NS32519 to L.I.
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