Melatonin, Apolipoprotein E And Alzheimer's Disease

Alzheimer's disease (AD) is a chronic and progressive brain disorder that resulting in the loss of cognitive functions primarily. Many studies have reported that ApoE genotype is associated with Alzheimer's disease (AD) and has a gene-dose effect on the risk of the disease. It has been reported that CSF melatonin levels in AD were much lower than aging control subjects. Melatonin shows a remarkable function including circadian rhythms, antiaging, immunomodulatory and antioxidant properties. It has been formerly considered that the pineal gland is the only source of melatonin. Circulating melatonin levels are derived primarily from the pineal gland while other sources of melatonin have also been reported. Previously, our results showed that the decreased CSF melaonin levels were associated with apoE genotype (ApoE3, ApoE4) and the CSF melatonin levels in AD patients expressing the ApoE-84/4 were significantly lower than those expressing ApoE-e3/4. In the present study, we firstly use neonatal (1-day-old Sprague-Dawley, SD) rats to investigate whether the cultured rat cortical astrocytes and the glioma C6 cell line can synthesize melatonin by cell culture, immunocytochemistry (ICC), western blotting and radioimmunoassay(RIA). In addition, we test whether the melatonin precursor (serotonin), the key enzymes (NAT and HIOMT) was found in the cultured rat cortical astrocytes and the glioma C6 cell line. Previous studies have demonstrated that melatonin plays an important role in the regulation of circadian rhythms under light/dark (LD) cycles. The rhythms in the in vivo rat pineal melatonin persists even under constant darkness (DD) conditions. In the present study, we investigate the possibility that melatonin synthesized by cultured rat cortical astrocytes may also show a diurnal rhythm. To investigate how ApoE genotype influences the NAT expression and astrocytes derived melatonin levels, we transfected the C6 cells with N1-EGFP, human ApoE2-EGFP, ApoE3-EGFP, and ApoE4-EGFP and selected stable cell lines expressing matched levels of ApoE2, ApoE3, and ApoE4. The results showed:1. In this study, a low amount of melatonin was for the first time found in cultures of rat cortical astrocytes and C6 cells. Although the level of melatonin produced in the rat cortical astrocytes was about two fifth of the rat pineal melatonin in the same culture conditions, cortical astrocyte secretion of melatonin will most probably contribute considerably to the high melatonin concentrations in the cerebrospinal fluid in vivo, considering the large amount of astrocytes in the brain.2. The presence of the melatonin precursor, serotonin, the enzyme NAT and the last enzyme HIOMT in the cultured rat cortical astrocytes show that the rat cortical astrocytes are able to synthesize melatonin. In addition, it suggests that rat cortical astrocytes synthesize melatonin via the traditional synthetic pathway. These findings indicate that in addition to pineal gland, rat astrocytes are an important physiological source of melatonin and that melatonin synthesized by astrocytes could somehow perform a protective function, possibly by acting as an intracrine, autocrine, or paracrine substance.3. In the present study, we investigated that the cultured rat cortical astrocytes had no diurnal rhythm. Statistical analysis suggested that there was no significant difference between day-time and night-time levels of melatonin in the cultured rat cortical astrocytes. The data rather suggest a role of the melatonin from astrocytes in trophic support to neurons or modulation of synaptic activity.4. The results suggested that ApoE protein levels was similar in different stably transfected cells (ApoE2, ApoE3, and ApoE4) by immunocytochemistry (ICC) and western blotting. The statistical analysis indicated that there was no significant difference in ApoE protein levels among the different stably transfected cells. The western blotting results and the statistical analysis suggested that the NAT expression in stable ApoE3 cells is much lower than those in ApoE2, ApoE4, and the control subjects and there were significant differences in NAT protein levels between stable ApoE3 cells and each of the ApoE2, ApoE4, and the control subjects. Furthermore, there is also significant difference in NAT protein expression between stable ApoE2 cells and ApoE4 cells. However, there was no significant difference in NAT protein levels between stable ApoE2 cells and N1 cells, between stable ApoE4 cells and N1 cells.