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. 1997 Mar 1;17(5):1683-90.
doi: 10.1523/JNEUROSCI.17-05-01683.1997.

Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide

M A Pappolla  1 , M Sos, R A Omar, R J Bick, D L Hickson-Bick, R J Reiter, S Efthimiopoulos, N K Robakis
Affiliations

Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide

M A Pappolla et al. J Neurosci. .

Abstract

Studies from several laboratories have generated evidence suggesting that oxidative stress is involved in the pathogenesis of Alzheimer's disease (AD). The finding that the amyloid beta protein (Abeta) has neurotoxic properties and that such effects are, in part, mediated by free radicals has provided insights into mechanisms of cell death in AD and an avenue to explore new therapeutic approaches. In this study we demonstrate that melatonin, a pineal hormone with recently established antioxidant properties, is remarkably effective in preventing death of cultured neuroblastoma cells as well as oxidative damage and intracellular Ca2+ increases induced by a cytotoxic fragment of Abeta. The effects of melatonin were extremely reproducible and corroborated by multiple quantitative methods, including cell viability studies by confocal laser microscopy, electron microscopy, and measurements of intracellular calcium levels. The importance of this finding is that, in contrast to conventional antioxidants, melatonin has a proposed physiological role in the aging process. Secretion levels of this hormone are decreased in aging and more severely reduced in AD. The reported phenomenon may be of therapeutic relevance in AD.

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Figures

Fig. 1.
Fig. 1.
Melatonin prevents death of N2a exposed to Aβ(25–35). N2a cells were plated and after 24 hr, during exponential growth phase, were treated with either scrambled peptide (control), adriamycin (control for apoptotic cell death; Marin et al., 1996), 50 μm Aβ(25–35), or 50 μm Aβ(25–35) with 10 μm melatonin for an additional 24 hr. Live cells were assessed by their fluorescence with BODIPY green (also see Fig. 2,right panels). Results are reported as means ± SD of four experiments (2 duplicate experiments on different days; minimum 100 cells studied per plate). *Measurements significantly different from control (p ≤ 0.02, pairedt test).
Fig. 2.
Fig. 2.
Representative confocal scanning images of annexin/propidium iodide and BODIPY green studies. The right panels illustrate representative images of one of the experiments plotted in Figure 1. Cultured N2a cells were exposed for 24 hr to either scrambled peptide (1), 50 μmAβ(25–35) (2), or 50 μm Aβ(25–35) plus 10 μm melatonin (3). After exposure to Aβ(25–35) alone (2), many cells showed a marked decrease in fluorescent intensity with BODIPY green, reflecting decreased cell viability (compare with panels 1 and3, which illustrate the fluorescent intensity exhibited by a similar number of cells exposed to either scrambled peptide alone in panel 1 or Aβ plus melatonin in panel 3). The areas photographed are representative fields of typical responses (magnification 1000×). A–C,Left, Representative images obtained from cells exposed to Aβ(25–35) and then stained by a dual fluorescent-tagging method with the probes annexin V-FITC (red) and propidium iodide (green). After examination with the appropriate filters, we counted the number of cells that stained simultaneously with both markers (necrosis) or with annexin V only (B or C, apoptosis). Exposure of cells to 50 μm Aβ(25–35) was followed by an almost exclusive increase in the number cells exhibiting red fluorescence only (annexin V), such as those illustrated in B and C. By 24 hr, 70 ± 25% of the cells exposed to Aβ(25–35) developed strong annexin (red) fluorescence and no increase in propidium iodide (green) fluorescence (means ± SD represent 2 duplicate experiments on different days, 4 experiments total; minimum 300 cells/plate counted). Such effects were prevented by simultaneous addition of melatonin to the culture medium [at 24 hr we counted 20 ± 10% annexin-positive cells in plates containing Aβ(25–35) plus melatonin and 15 ± 10% annexin-positive cells in control plates containing scrambled peptide alone].
Fig. 5.
Fig. 5.
Cell viability after exposure to Aβ alone or Aβ with various concentrations of melatonin or PBN. N2a cells were plated and after 24 hr, during exponential dividing phase, exposed to the indicated concentrations of Aβ(25–35) for 6 hr and treated with either melatonin or PBN at the indicated concentrations. These experiments were performed at 6 hr because cell death was readily apparent by this time. Viable cells are expressed as a percentage of controls and assessed by their ability to exclude trypan blue. Similar dose– responses were obtained by BODIPY green fluorescence (data not shown). Differences in survival between cells exposed to Aβ alone versus Aβ with melatonin were statistically significant for all concentrations of Aβ and melatonin (i.e., 50 μm Aβ vs 50 μm Aβ + 1.2 μm melatonin, p < 0.002; 50 μm Aβ vs 50 μm Aβ + 10 μmmelatonin, p < 0.001).
Fig. 3.
Fig. 3.
Electron microscopy of N2a cells exposed to Aβ(25–35). Scanning electron microphotographs illustrate conspicuous cell retraction (A, B) induced by the amyloid peptide. Note marked membrane blebbing (B).C, D, Transmission electron microscopy preparations depict chromatin misdistribution and karyorrhexis in cultured N2a induced by Aβ. The prevalence of membrane blebbing (defined as the percentage of cells exhibiting diffuse involvement by large and small blebs on more than one-half of their surface) and cell retraction was quantitated by counting 150 cells per scanning preparation (total of 4 experiments per condition; see Table 1).
Fig. 4.
Fig. 4.
Experiments on PC12 cells exposed to Aβ(25–35) (A) and N2a and PC12 cells exposed to Aβ(1–40) (B and C, respectively). In these experiments cells were plated as described in the previous experiments, except that PC12 cells required 4 d of growth on collagen-coated plates. Cells were exposed to 50 μm Aβ(25–35) (A) or 100 μm Aβ(1–40) (B, C) for 24 hr. Melatonin, where indicated, was at 50 μm. Values represent the means ± SD of four experiments; a minimum of 500 cells was counted per culture plate. Cell viability was assessed by trypan blue exclusion and expressed as a percentage of controls.
Fig. 6.
Fig. 6.
Lipid peroxidation induced by Aβ(25–35) is prevented by melatonin or PBN. The byproduct malondialdehyde acid (MDA) was measured in N2a cell lysates as described (Omar et al., 1987) at the indicated concentrations of melatonin (Fig. 5A), PBN (Fig. 5B), and Aβ(25–35). Values are the means of three determinations. SE in all measurements was <20% of the mean. Cells were exposed to Aβ(25–35) for 24 hr with and without melatonin or PBN.
Fig. 7.
Fig. 7.
Melatonin prevents cell death induced by inhibition of SOD. Cells were plated as previously noted in Figure 1and exposed to DDTC for 24 hr at the indicated concentrations. Melatonin was added at the stated concentrations. Survival was determined by the trypan blue exclusion method and expressed as percentage of controls (no DDTC). Data represent the means ± SD for four experiments (2 duplicated experiments on different days).
Fig. 8.
Fig. 8.
Time course study of fluo-3 fluorescence increase induced by Aβ(25–35) and prevention by melatonin. Cells were exposed to 50 μm scrambled peptide (control), 50 μmAβ(25–35), or 50 μm Aβ(25–35) plus 5 μm melatonin. Aβ alone was significantly different from control and Aβ plus melatonin after 6 hr at all time points (p < 0.002). There were no significant differences between control and Aβ plus melatonin at any time point.
Fig. 9.
Fig. 9.
Melatonin prevents intracellular Ca2+increases induced by Aβ. Representative images from various experimental conditions illustrate the characteristic fluorescent patterns exhibited by fluo-3 in cells after 12 hr exposure to 50 μm Aβ(25–35) plus 5 μm melatonin (A), 50 μm Aβ(25–35) (B), 0.03 μg/ml adriamycin (C), and 50 μm scrambled peptide (D). (Final magnifications are 2000× for A and B and 3000× for C and D.)
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