Melatonin Treatment Enhances Aβ Lymphatic Clearance in a Transgenic Mouse Model of Amyloidosis

Abstract

Background: It has been postulated that inadequate clearance of the amyloid β protein (Aβ) plays an important role in the accumulation of Aβ in sporadic late onset Alzheimer's disease (AD). While the blood brain barrier (BBB) has taken the center stage in processes involving Aβ clearance, little information is available about the role of the lymphatic system. We previously reported that Aβ is cleared through the lymphatic system. We now assessed lymphatic Aβ clearance by treating a mouse model of AD amyloidosis with melatonin, an Aβ aggregation inhibitor and immuno-regulatory neurohormone.

Objective: To confirm and expand our initial finding that Aβ is cleared through the lymphatic system. Lymphatic clearance of metabolic and cellular "waste" products from the brain into the peripheral lymphatic system has been known for a long time. However, except for our prior report, there is no additional experimental data published about Aβ being cleared into peripheral lymph nodes.

Methods: For these experiments, we used a transgenic mouse model (Tg2576) that over-expresses a mutant form of the Aβ precursor protein (APP) in the brain. We examined levels of Aβ in plasma and in lymph nodes of transgenic mice as surrogate markers of vascular and lymphatic clearance, respectively. Aβ levels were also measured in the brain and in multiple tissues.

Results: Clearance of Aβ peptides through the lymphatic system was confirmed in this study. Treatment with melatonin led to the following changes: 1-A statistically significant increase in soluble monomeric Aβ40 and an increasing trend in Aβ42 in cervical and axillary lymph nodes of treated mice. 2- Statistically significant decreases in oligomeric Aβ40 and a decreasing trend Aβ42 in the brain.

Conclusion: The data expands on our prior report that the lymphatic system participates in Aβ clearance from the brain. We propose that abnormalities in Aβ clearance through the lymphatic system may contribute to the development of cerebral amyloidosis. Melatonin and related indole molecules (i.e., indole- 3-propionic acid) are known to inhibit Aβ aggregation although they do not reverse aggregated Aβ or amyloid fibrils. Therefore, these substances should be further explored in prevention trials for delaying the onset of cognitive impairment in high risk populations.

Keywords: Alzheimer's disease; amyloid clearance; beta amyloid; lymphatic nodes; melatonin; transgenic mice..

Figure 1.

Saline soluble monomeric Aβ40 in control and melatonin treated mice. Brain and lymph node homogenates in saline along with plasma (B) from Tg2576 mice were evaluated by a sensitive and specific ELISA assay for Aβ40. The n values for control (Con) and melatonin (Mel)-treated samples from 4 and 15.5 month-old animals are listed in order for lymph node (n=6, 6, 15, 15); plasma (n=2, 5, 14, 28) and brain (n=12, 14, 13, 14); Aβ40 oligomers measurements in brain at 15.5 age months are also shown in controls and after treatment with melatonin (n= 8, 16). Con = Control mice. Mel = Melatonin treated mice. Since acceptable and consistent Aβ oligomer standards are not available to establish curves, we have compared the relative luminescence values detected in brain extracts from control and melatonin-treated mice. Oligomeric Aβ40 was not detectable in plasma or in lymph nodes (not shown). Some of the untreated control groups have lesser numbers of animals than the treated groups, mostly reflecting premature deaths (known attrition rate in AD transgenics) and to a lesser degree, to inadvertent tissue/sample loss. Melatonin dramatically increased survival in the treated groups to levels comparable to non-transgenic mice (not shown).

FIGURE 2.

Saline soluble monomeric Aβ42 in control and melatonin treated mice. Brain and lymph node homogenates in saline along with plasma (B) from Tg2576 mice were evaluated by a sensitive and specific ELISA assay as shown for figure 1. The n values for control (Con) and melatonin (Mel)-treated samples from 4 and 15.5 month-old animals are listed in order for lymph node (n=3, 6, 16, 16); plasma (n=3, 5, 14, 28) and brain (n=3, 2, 13, 14). Aβ40 oligomers measurements in brain at 15.5 age months are also shown in controls and after treatment with melatonin (n= 8; 15). As shown in figure 1, Aβ42 oligomers measurements in brain are shown at 15.5 age months in controls and after treatment with melatonin (n=8; 15). Con = Control mice. Mel = Melatonin treated mice. As mentioned for figure 1, acceptable and consistent Aβ42 oligomer standards are not available to establish curves; thus, we have compared the relative luminescence values detected in brain extracts from control and melatonin-treated mice. As with Aβ40, oligomeric Aβ42 was not detectable in plasma or in lymph nodes.