Effects of melatonin and age on gene expression in mouse CNS using microarray analysis

Abstract

The expression levels of a number of genes associated with inflammation and immune function change with advancing age. Melatonin modulates gene expression levels of several of these genes. Therefore the declining levels of melatonin associated with age may play a role in the physiological effects of aging. We used oligonucleotide microarrays to measure age-related changes in mRNA expression in the murine CNS, and to study the effect of prolonged administration of dietary melatonin upon these changes. CB6F1 male mice were fed 40 ppm melatonin for 2.1 months prior to sacrifice at age 26.5 months, and compared with both age-matched controls and young, 4.5-month-old untreated controls. Total RNA was extracted from whole brain (excluding cerebellum and brain stem) and individual samples were hybridized to Affymetrix Mouse 430-2.0 arrays. The expression of a substantial number of genes was modulated by melatonin treatment and changes in selected genes were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A subset of these genes did not change with age. Conversely, some genes modulated by age were also modulated by melatonin treatment. In general, melatonin treatment drove the expression levels of these genes closer to the expression levels detected in the younger animals. Notably, the abundance of lipocalin 2 (Lcn2) mRNA increased with age and was decreased in old animals treated with melatonin. Lcn2 is a member of the acute phase response family of proteins and its mRNA levels in the brain increase in response to inflammation. Many of the genes with expression reduced by melatonin are involved in inflammation and the immune system. This suggests that melatonin treatment may influence the inflammatory responses of old animals, driving them to resemble more closely those occurring in young animals.

Introduction

Markers of chronic inflammation and oxidative damage increase in the mammalian CNS with age, whereas melatonin has demonstrated antioxidant, anti-inflammatory and immunomodulatory properties (Zhang et al., 1997, Reiter et al., 2000, Sharman et al., 2002a, Sharman et al., 2002b, Sharman et al., 2004, Pei and Cheung, 2004). The pulsatile nocturnal peak level of melatonin decreases with age in rats (Pang et al., 1990), mice (Lahiri et al., 2004a) and humans (Tozawa et al., 2003), and is predicted to be involved in regulation of sleep, which is commonly disturbed in the elderly (Foley et al., 1995).

The only well-established means of lengthening the lifespan of animals is caloric restriction, and notably, short-term caloric restriction increases melatonin levels in brain and gut (Bubenik et al., 1992). Moreover, dietary supplementation with melatonin increases the lifespan of mice (Pierpaoli and Regelson, 1994, Anisimov et al., 2000) and rats (Oaknin-Bendahan et al., 1995). Thus it is reasonable to conclude that melatonin may play a role in promoting life extension. However, little is known of the mechanisms by which melatonin exerts these effects.

Melatonin is secreted by the pineal gland, which in mice is located atop the cerebral cortex but outside the blood–brain barrier. Melatonin receptor mRNA is detected in a number of areas within the CNS, including the pituitary pars tuberalis, the suprachiasmatic nucleus of the hypothalamus (von Gall et al., 2002), the hippocampus and cerebellum (Al-Ghoul et al., 1998). In the rat, melatonin receptor density decreases with age in the hypothalamus and the hippocampus (Laudon et al., 1988), and melatonin supplementation increases both melatonin levels (Menendez-Pelaez et al., 1993, Lahiri et al., 2004a) and melatonin binding (Oaknin-Bendahan et al., 1995) in the brains of older animals. In the current study we sought to gain a better understanding of melatonin's possible neuroprotective mechanisms by comparing the age-related CNS gene expression patterns of young and old mice and old mice receiving dietary melatonin. We sought to improve the understanding of the aging process in the brain generally; thus we measured gene expression changes related to aging and melatonin common to the cerebrum as a whole, rather than inquire into detailed changes occurring in specific regions.