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Keywords:

  • Alzheimer;
  • β-amyloid;
  • dementia;
  • melatonin;
  • oxidative stress;
  • protein nitration

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Increased levels of a 40–42 amino-acid peptide called the amyloid β protein (Aβ) and evidence of oxidative damage are early neuropathological markers of Alzheimer's disease (AD). Previous investigations have demonstrated that melatonin is decreased during the aging process and that patients with AD have more profound reductions of this hormone. It has also been recently shown that melatonin protects neuronal cells from Aβ-mediated oxidative damage and inhibits the formation of amyloid fibrils in vitro. However, a direct relationship between melatonin and the biochemical pathology of AD had not been demonstrated. We used a transgenic mouse model of Alzheimer's amyloidosis and monitored over time the effects of administering melatonin on brain levels of Aβ, abnormal protein nitration, and survival of the mice. We report here that administration of melatonin partially inhibited the expected time-dependent elevation of β-amyloid, reduced abnormal nitration of proteins, and increased survival in the treated transgenic mice. These findings may bear relevance to the pathogenesis and therapy of AD.

Abbreviations used
AD

Alzheimer's disease

amyloid β protein

BA27

anti-Aβ1–40 antibodies

BC05

anti-Aβ1–42 antibodies

NT

nitrotyrosine

Many of the advances in Alzheimer's disease (AD) have followed the identification of a 40–42 amino-acid peptide called the amyloid β protein (Aβ) as the essential pathologic marker of the disease (Glenner and Wong 1984; Masters et al. 1985). Brains with AD show extensive accumulation of amyloid fibrils, mostly within senile plaques and in the cerebral and meningeal vasculature (Esiri et al. 1997). Aβ is the product of various proteolytic cleavages of a larger protein called the amyloid precursor protein or AβPP (Neve and Robakis 1998). Under a number of in vitro experimental conditions, Aβ aggregates spontaneously into insoluble amyloid fibrils which are virtually indistinguishable from the amyloid that accumulates in AD. Misprocessing of AβPP or inadequate clearance of Aβ peptides are both postulated to play important roles in amyloid accumulation (Neve and Robakis 1998; Selkoe 1998); however, the mechanisms underlying amyloid formation and neuronal degeneration in the human brain are unknown. It has been proposed by many investigators that an increased level of Aβ leads to widespread neuronal degeneration (Neve and Robakis 1998). Aβ appears to promote neuronal death, at least in part, by generating a high level of several oxygen free radicals (Esiri et al. 1997) a process that appears dependent on the predominant β-sheet conformation of the Aβ protein (Pappolla et al. 1998). Previous investigations suggest that levels of soluble Aβ become elevated in the brain very early in AD and that these increases correlate with declines in cognitive functions (Naslund et al. 2000). Whether oxidative stress is a cause or consequence of amyloid deposition is not without dispute; recent studies suggest that oxidative damage contributes to AD pathogenesis before the onset of Aβ accumulation in the AD brain (Nunomura et al. 2001; Pratico et al. 2001). Despite these controversies, it is generally accepted that reducing amyloid formation or enhancing neuronal survival are among the most important therapeutic targets in AD.

Numerous studies have shown that the pineal hormone melatonin is decreased during the aging process (Reiter 1992) and that patients with AD have more profound reductions of this substance (Souetre et al. 1989; Mishima et al. 1994; Liu et al. 1999).

Melatonin has demonstrated neuroprotective and antioxidant properties against Aβ mediated oxidative injury in vitro (Bozner et al. 1997; Pappolla et al. 1997). In addition, it has been shown that melatonin inhibits the formation of β-sheets and amyloid fibrils (Pappolla et al. 1998; Poeggeler et al. 2001). However, the essential question of whether there is a direct relationship between melatonin and the biochemical pathology of AD has not been addressed. The recent availability of transgenic models of amyloidosis provided us with an opportunity to begin addressing this question. Therefore, we studied the effects of administering melatonin to a transgenic mouse model of AD on the age-related increase of Aβ peptides, abnormal protein nitration and survival. Melatonin treatment inhibited the development of these disease markers and virtually eliminated the excessive mortality affecting the transgenic mice.

Melatonin treatment of AD transgenic mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

For this study, we used the transgenic mice Tg2576 that over-express the 695-amino-acid isoform of human AβPP containing a Lys670 [RIGHTWARDS ARROW] Asn, Met671 [RIGHTWARDS ARROW] Leu mutation found in a Swedish family with early onset AD. In these animals, levels of Aβ begin to rise in the brain by 6 months of age and by 9–10 months they develop senile plaque-like deposits of Aβ (Hsiao et al. 1996). All animals were genotyped twice, at birth and after death, using a standard PCR protocol (Hsiao et al. 1995). Mice in each experimental group were housed up to 4 to a cage in air-conditioned rooms at 22°C and fed ad libitum with AIN76A (Bethlehem, PA, USA). The Institutional Animal Review Board approved the use of mice for this study and national guidelines for humane treatment were followed.

Ultrapure melatonin (99.9%) was purchased from Helsinn Pharmaceuticals (Biasca, Switzerland). All other chemicals were purchased from Sigma (St Louis, MO, USA). Melatonin was dissolved in ethyl alcohol (absolute) at a concentration of 50 mg/mL as a stock solution and then diluted in the drinking water to a final concentration of 0.5 mg/mL. An equal amount of solvent stock solution without melatonin was added to the drinking water of untreated mice. Treated and untreated animals consumed similar amounts of fluid (an average of 3 mL/day) as estimated by periodic body weights and solution measurements. The treatment was initiated at 4 months of age and was continued up to various time points (8, 9.5, 11 and 15.5 months) at which the animals were killed. Brain tissue melatonin in treated and untreated mice was measured by an HPLC method, using a published protocol that maximizes tissue extraction of the hormone (Poeggeler and Hardeland 1994; Barlow-Walden et al. 1995). The number of animals in each group is shown in Table 1.

Table 1.  Cohorts of untreated (controls) and melatonin-treated mice
Untreated transgenic groupsMelatonin-treated groups
Age (mo)40 SDS (n)42 SDS (n) 40 FA (n)42 FA (n)Age (mo)40 SDS (n)42 SDS (n)40 FA (n)42 FA (n)
  1. 40, Aβx-40; 42, Aβx-42; SDS, 2% sodium dodecyl sulfate extracted fractions; FA, 70% formic acid extracted fractions; mo, age in months; n, number of mice.

15.5666615.56666
11.0777711.07777
9.566669.57758
8.044448.04434

Aβ measurements in brain tissue

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Using highly specific antibodies and a very sensitive sandwich enzyme linked immunoassay (ELISA) we quantified soluble and insoluble Aβ40 and Aβ42 in brain homogenate fractions extracted with 2% sodium dodecyl sulfate (SDS) and 70% formic acid, respectively. The standardization, sensitivity, and specificity of the method have been described in a previous study (Matsubara et al. 1999). Similar frozen hemispheric slices of brain tissue, matched for the areas sampled, were obtained from each animal and homogenized with a motor-driven Teflon/glass homogenizer (20 strokes) in 1 mL of 2% SDS and then centrifuged at 100 000 g for 1 h using a Hitachi (Ibaraki, Japan) RP100AT rotor. To quantitate the Aβ species, the resultant supernatants (soluble fractions) and the pellets (insoluble fractions) were subjected to a sensitive sandwich ELISA as described (Suzuki et al. 1994; Asami-Odaka et al. 1995; Matsubara et al. 1999). Briefly, microplates (Immunoplate I, Nunc, Rockilde, Denmark) were pre-coated with anti-Aβ monoclonal antibody BNT77 that recognizes both Aβ1–40 and 1–42 and is specific for residues 11–16 of Aβ and then incubated for 24 h at 4°C (100 μL/well). Plates were washed and incubated for 24 h at 4°C with either specific anti-Aβ1–40 antibodies (BA27) or specific anti-Aβ1–42 antibodies (BC05, specific against the Aβ35–43 domain) conjugated with horseradish-peroxidase. The color reaction was developed with 3,3′,5,5′-tetramethylbenzidine and measured at 450 nm on a microplate Reader (Molecular Devices, CA, USA). Synthetic Aβ1–40 and Aβ1–42 (Sigma) of known concentrations (calculated by amino-acid composition) were used as standards. The obtained values were corrected with the wet weight of each tissue sample and expressed as pmol/gram. For quantification of soluble Aβ1–40 and Aβ1–42, we used the 2% SDS supernatants which were diluted 1 : 400 or 1 : 800 with 20 mm phosphate buffer (pH 7.2) containing 0.4 m NaCl, 2 mm EDTA, 10% Block Ace (Dainippon, Japan), 0.2% bovine serum albumin, and 0.05% NaN3 (buffer EC). For quantification of the insoluble fractions, the pellet was washed once, and then extracted further with 1 mL of 70% formic acid. The insoluble 70% formic acid extracts, were neutralized with 1 m Tris-HCl (pH 8.0) at a dilution of 1 : 1000. The homogenate was then centrifuged at 100 000 g for 1 h and the resultant supernatant (corresponding to the insoluble fraction) was used for quantification.

Quantitative immunoblots of AβPP levels

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Brain areas (right frontal pole) were homogenized at a w/v ratio of 10–1 in 100 mm Tris pH 8.0, 150 mm NaCl, 1% SDS containing protease inhibitors (Complete, Boehringer Mannheim). Fifty micrograms of protein per sample were loaded on 10% Tricine gels and proteins were transferred by electrophoresis to Immobilon filters (Millipore Corporation, Bedford, MA, USA). The filters were stained with an anti-AβPP antibody (KS7) and an anti-actin antibody, followed by a horseradish peroxidase conjugated either an anti-rabbit or an anti-mouse antibody. The anti-AβPP antibody is a polyclonal directed against the C-terminal fragment of AβPP. Characterization of this antibody has been published (Sambamurti et al. 1999). The anti-actin antibody was a monoclonal obtained from a commercial source (Sigma, A 4700). Bands were quantitated by chemiluminescent detection using ECL Plus (Amersham) and phosphorimage analysis (Molecular Dynamics, Sunnyvale, CA, USA; Storm 840).

Nitrotyrosine measurements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Brain tissue nitrotyrosine (NT) in treated and untreated mice was measured by an HPLC method (Strand et al. 2000) using a Hewlett-Packard P 1100 series HPLC and a 25-mm × 4.6 mm C18 HPLC column (Beckman Ultrasphere ODS). The assay consists of an isocratic ion-pair 12 min run followed by a sharp gradient to 100% acetonitrile. The UV detector was set to monitor at 355 ± 16 nm and 280 ± 4 nm with a background subtract function of 550 ± 100 nm. The peak areas were plotted against standards and unknowns were read off standard curves and quantified. Samples analyzed for free NT were injected following de-proteination by ultrafiltration. For protein associated NT, the samples were injected after proteolysis and ultrafiltration.

Immunohistochemistry and image analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Immunohistochemistry and image analysis were performed using published methods (Refolo et al. 2000). The 4G8 antibody was used for tissue detection of Aβ deposition after formic acid treatment. Antibody dilution was 1 : 1000. Quantification of amyloid load was performed by image analysis using a CoolSNAP Camera and Image-Pro Plus Software (Media Cybernetics, Silver Spring, MD, USA). Measurements were performed at 15.5 months of age.

Survival studies

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Several studies have emphasized the significantly shortened life expectancy and high mortality observed in AD patients, a fact reflected in transgenic mice (Wolfson et al. 2001). We analyzed the survival experience of melatonin-treated and -untreated transgenics from a separate on-going experiment. At the onset of this experiment, there were 44 treated and 44 untreated mice, which began receiving melatonin at 4 months of age with melatonin being administered in the drinking water at a dose of 2 mg/mL suspended in a 5% hydroxypropyl β-cyclodextrin solution. Untreated mice received vehicle without melatonin. Survival of treated and untreated mice was determined using Kaplan-Meier plots (StatXact 4 for Windows, Cytel Software, Cambridge, MA, USA; 1999). The null hypothesis on the survival experience of the two groups was tested using the Generalized Wilcoxon-Gehan test (StatXact 4 for Windows, Cytel Software; 1999).

Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Figure 1 and Table 2 illustrate the measured levels of total brain Aβ (formic acid + SDS fractions, Fig. 1) and of two different Aβ species (formic acid and SDS fractions, Table 2), obtained from melatonin-treated and -untreated transgenic mice at each of the studied time points. As it can be appreciated, marked and statistically significant differences (indicated by asterisks) were observed. The dose of melatonin used in this study resulted in increased brain tissue concentrations of melatonin (Fig. 2). As previously reported, there was substantial variation in brain melatonin levels among treated mice (particularly in older animals) most likely resulting from prolonged melatonin administration which causes enhanced liver clearance and variability in gastrointestinal absorption (Hendler and Rorvik 2001).

image

Figure 1. Aβ peptide levels in treated and untreated transgenic mice. The values shown at each time point represent the mean with 1 SE indicated. The p-values (two-tailed student t-test) are noted above each bar in the graphs. The values of total brain Aβ represent the sum of the four Aβ fractions (40 SDS, 42 SDS, 40 FA, 42 FA).

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Table 2.  Aβ concentrations for untreated and melatonin-treated mice
AgeGroup40 SDS42 SDS40 FA42 FA
Aβ levelchange Aβ levelchange Aβ levelchange Aβ levelchange
p-valuep-valuep-valuep-value
  • *

    Marked and statistically significant differences; U, untreated; T, treated; 40, Aβx-40; 42, Aβx-42; SDS, 2% sodium dodecyl sulfate extracted fractions; FA, 70% formic acid extracted fractions; Age, age in months; change, the percentage of concentration (pmol/g brain) change in the corresponding Aβ species caused by treatment with melatonin.

8.0U8.7 ± 0.8+ 34%1.0 ± 0.9− 82%20.2 ± 9.5− 85%1.3 ± 0.2− 94%
T11.7 ± 2.50.3020.2 ± 0.10.3663.0 ± 0.80.374 0.1 ± 0.00.001*
9.5U26.8 ± 7.2− 49%2.3 ± 1.5− 80%34.7 ± 8.1− 70%2.5 ± 0.9− 98%
T13.6 ± 4.40.1340.5 ± 0.30.23210.3 ± 7.30.1130.1 ± 0.00.007*
11.0U49.5 ± 6.0− 40%3.7 ± 1.2− 82%97.2 ± 14.8− 73%25.9 ± 8.3− 90%
T29.5 ± 5.90.035*0.7 ± 0.40.037*26.2 ± 9.10.003*2.5 ± 1.40.016*
15.5U920 ± 209− 77%86.3 ± 6.4− 37%3568 ± 542− 67%406 ± 77− 70%
T208 ± 220.007*54 ± 180.1251183 ± 2540.005*122 ± 490.011*
image

Figure 2. Melatonin levels measured in brain tissue of treated and untreated mice. The values, as determined by an HPLC method, are shown at each time point with 1 SE indicated. The p-values (two-tailed student t-test) are noted above each bar in the graphs.

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Melatonin treatment does not cause changes in AβPP expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Previous investigations demonstrated that, at the doses used in this study, melatonin is devoid of toxicity and is neuroprotective against various forms of injury in the CNS (Reiter et al. 2000). However, it was prudent to exclude the possibility that melatonin could have had neurotoxic effects at the given dose, leading to a reduction in AβPP expression in the treated mice. Quantitative western blots from brain tissue showed no differences in the expression of AβPP holoprotein between treated and untreated animals (Fig. 3), suggesting that the reduction in Aβ levels in treated mice was not the result of decreased AβPP expression.

image

Figure 3. AβPP and actin western blots. Quantitative immunoblots between melatonin-treated and untreated mice revealed no significant differences in AβPP levels. Genotypes are denoted by +, transgenic and –, non-transgenic.

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Melatonin treatment reduces abnormal protein nitration in transgenic mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

Administration of melatonin was followed by a significant reduction of protein nitration in brain tissue of mice. Figure 4 shows the levels obtained in the treated and untreated transgenics.

image

Figure 4. Protein nitration in melatonin treated versus untreated. A consistent reduction of protein nitration was observed in treated versus untreated transgenics. The values shown here denote the mean nitration for each group with 1 SE indicated, p-values represent one-tailed unpaired t-tests.

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Amyloid load in melatonin treated and untreated transgenic mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

In contrast with the conspicuous reductions observed in levels of Aβ as measured by ELISA, there was only a trend without reaching significance in amyloid load between treated and untreated transgenic mice (Fig. 5). However, the variability of the data for the sample size was so large at 15.5 months (the time point when a sufficient number of plaques can be visualized) that the difference between the groups could not be estimated with statistical precision. Using a Student's t-test (Systat 9.0) the mean difference (untreated minus treated) between the two groups was 1.54; the 0.95 confidence interval is (− 5.39, 8.47). Because the attributes of the data were not consistent with the assumptions required for the validity of the Student's t-test, we used an exact Hodges-Lehmann test (StatXact 4.0; Cytel, Cambridge, MA, USA). A Bayesian model (Confidence Profile software; Academic Press Inc., Chestnut Hill, MA, USA) of this data gives a posterior probability of 0.66 that the difference is greater than zero (Fig. 5). Although this may reflect the fact that the dose of melatonin used was insufficient to fully overcome the effect of the mutant gene as the age of the mice advanced, there may be other potential explanations for the discrepancy between the biochemical and morphometric measurements (see Discussion).

image

Figure 5. Amyloid load in treated and untreated mice. Means of the amyloid load for the treated and untreated mice as determined by image analysis are illustrated in the bar graph. There were six animals allocated to each group. The right panel depicts the posterior probability of 0.66 that the difference (untreated minus treated) is greater than zero.

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Survival studies

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

In groups of 44 treated mice and 44 untreated transgenics (from an ongoing experiment), we found that the survival of transgenic mice receiving melatonin was strikingly enhanced (p = 0.002). To the time of this writing, the survival data for the untreated group consisted of 13 deaths and 31 survivals while the treated group consisted of 3 deaths and 41 survivals. The respective Kaplan–Meier plot is shown in Fig. 6.

image

Figure 6. Survival of treated versus untreated mice. Marked differences in mortality between treated and untreated transgenic mice were observed in mice from ongoing experiments, as shown in the respective Kaplan–Meier plots. The p-value (two-tailed student t-test) is noted on the graph.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References

The data presented in this study showed that melatonin administration to AD transgenic mice is associated with a reduction in a number of important markers of the disease including Aβ levels, protein nitration and survival.

Significant reductions in Aβ levels, as detected by ELISA methods, may reflect the anti-fibrillogenic activity of melatonin. Previous studies using circular dichroism, Fourier transform infra-red spectroscopy and electron microscopy have demonstrated that melatonin has inhibitory effects on the formation of secondary β-sheet structures and amyloid fibril formation in vitro (Pappolla et al. 1998; Poeggeler et al. 2001). Using nuclear magnetic resonance spectroscopy, we have previously shown that melatonin disrupts the imidazole-carboxylate salt bridges between the side chains of the His+ and Asp residues in Aβ peptides (Pappolla et al. 1998) that are important for the formation and stabilization of β-sheet structures and formation of amyloid fibrils. It is therefore possible that by maintaining Aβ in a more protease sensitive conformation, melatonin may facilitate removal of Aβ from the brain resulting in the measured decreases in Aβ levels in the treated mice.

In sharp contrast with the impressive reduction in Aβ levels as measured by ELISA, there was only a trend but no significant differences in amyloid deposition at 15.5 months of age as shown by quantitative immunohistochemistry. Although there was a high variability in the number of plaques at the age studied, it is also possible that the treatment became insufficient to overcome the effects of the mutant gene as the mice became older. While this may be a consequence of a suboptimal dose of melatonin at 15.5 months (the mean melatonin concentration obtained in the brains of the treated mice was approximately half of that needed for optimal inhibition of Aβ fibrillogenesis in vitro; Pappolla et al. 1998), there may be additional reasons for the difference. One possibility is that the length of treatment resulted in reduced therapeutic efficacy. Melatonin clearance occurs by the hepatic cytochrome P450 system and its high first liver pass contributes to the increased variability in brain melatonin levels (Ursing et al. 2002). In this regard, the role of the brain cytochrome system pertaining to melatonin clearance is currently unknown. In addition, we do not know whether the sites of interaction between melatonin and Aβ may be blocked in vivo. Because this was only a limited ‘proof of concept’ investigation, additional experiments with melatonin analogs are warranted. Other experimental design variables such as dose, sample size and mode of administration will also have to be considered in subsequent studies.

An important outcome of the treatment was the effect on protein nitration. Among several markers of neuronal damage, protein nitration of tyrosine residues, mediated by breakdown of peroxynitrate, has been postulated to be a major pathway for Aβ-mediated injury (Good et al. 1996; Smith et al. 1997). Melatonin administration caused a statistically significant reduction of this injury indicator. Interestingly, the effect of melatonin treatment on protein nitration was most robust at younger ages and decreased in the older animals, in conceptual agreement with the issues discussed previously pertaining to amyloid load.

Finally, one potentially important effect of the treatment was the striking difference on the survival between treated and untreated mice. It is well known that Alzheimer's disease shortens life expectancy; estimates of median survival after the onset of dementia have ranged from 3.1 to 9.3 years (Wolfson et al. 2001). Although the mortality in mice occurs relatively early in the murine disease, their death rates are reminiscent of the high mortality afflicting AD patients. It is therefore interesting that the treatment virtually eliminated the increased mortality observed in transgenic untreated mice. One caveat to be kept in mind is that the relationship between the mode of death in mice and in AD patients has not yet been determined.

Melatonin is a pineal hormone secreted during the dark phase of the circadian cycle (Reiter 1992). Its rhythmic synthesis and secretion decline sharply during the aging process (Iguchi et al. 1982; Touitou et al. 1984; Sack et al. 1986; Reiter 1992). Importantly, results from various studies have shown that patients with AD have more profound reductions of this hormone (Souetre et al. 1989; Mishima et al. 1994; Liu et al. 1999). These intriguing facts are in sharp contrast with various potential therapeutic approaches being developed for AD which are based on neuroprotective and/or anti-amyloidogenic strategies but have no physiological correlation with the aging process. Not only has a decreased melatonin level been repeatedly demonstrated in populations with dementia, a recent and puzzling observation shows that the lowest levels of the hormone occur in patients who are homozygous for the apoE-ε4/4 allele (Liu et al. 1999).

An interesting body of literature in the melatonin field suggests that melatonin plays an important regulatory role in cholesterol metabolism (Müller-Wieland et al. 1994); this raises interesting unexplored issues pertaining to AD pathogenesis, particularly in view of the emerging connection between cholesterolemia and amyloid pathology (Refolo et al. 2000). Further research is necessary to elucidate the mechanisms of in vivo reduction of Aβ levels and neuroprotection by melatonin. Melatonin has pleiotrophic properties (i.e. antioxidant, anti-amyloidogenic, copper chelating, modulation of cholesterol homeostasis, COX inhibition) and it is difficult to determine at this time the extent of the contribution from each of the mentioned properties to the overall effects of the treatment.

Most likely, however, the observed effects reflect the anti-aggregating properties of the hormone. Previous in vitro studies have shown that the optimal ratio for inhibition of Aβ aggregation is around 1 : 1 (pMol : pMol) (Pappolla et al. 1998); a similar ratio occurs under normal physiological conditions in youth, during the dark phase of the circadian cycle (Pang et al. 1974; Johnson-Wood et al. 1997). Another possibility is that melatonin causes inhibition of oxidative pathology that can in itself play a role on amyloid formation (Yan et al. 1996; Zhang et al. 1997). Using a wide variety of techniques such as electron spin resonance spectroscopy, pulse radiolysis and in vitro cell culture systems, melatonin has been shown to be an efficient scavenger of the hydroxyl radical (Pang et al. 1974; Tan et al. 1993; Poeggeler et al. 1994; Chyan et al. 1999), peroxynitrate (Gilad et al. 1997; Cuzzocrea et al. 1998), and singlet oxygen (Cagnoli et al. 1995; King and Scaiano 1997; Zang et al. 1998). All these highly reactive species have been implicated in AD (Pappolla et al. 2000).

It must be emphasized that this study is only a first step toward exploring the effects of melatonin in an in vivo model of AD and no claims are made about a potential physiological role of this hormone in the disease pathogenesis. A major unresolved question is whether physiological levels of the melatonin can contribute to the total antioxidant and/or neuroprotective capacity of the brain during sporadic age-associated human amyloidogenesis. In this study, as well as in the bulk of the research conducted thus far, pharmacological levels of melatonin were utilized. Whether a reduction in endogenous melatonin influences the onset and/or severity of amyloid deposition or the symptoms of AD is unknown. Available data from mutagenesis studies suggest that even miniscule (in the pM range) amounts of endogenous brain melatonin are sufficient to reduce the oxidative damage from mutagen exposure in vivo (Tan et al. 1993). Likewise, models of ischemia and excitotoxicity also support the concept that the endogenous levels of melatonin are sufficient for neuroprotection in vivo (Manev et al. 1996; Kilic et al. 1999). No controlled, double-blind studies are currently available concerning the preventive or therapeutic potential of melatonin in AD. The results presented here suggest that melatonin or related indole structures can be explored as disease-modifying agents in AD.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Melatonin treatment of AD transgenic mice
  5. Aβ measurements in brain tissue
  6. Quantitative immunoblots of AβPP levels
  7. Nitrotyrosine measurements
  8. Immunohistochemistry and image analysis
  9. Survival studies
  10. Results
  11. Melatonin treatment reduces Aβ levels in Tg2576 transgenic mice
  12. Melatonin treatment does not cause changes in AβPP expression
  13. Melatonin treatment reduces abnormal protein nitration in transgenic mice
  14. Amyloid load in melatonin treated and untreated transgenic mice
  15. Survival studies
  16. Discussion
  17. Acknowledgements
  18. References
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