Alterations of Clock Gene RNA Expression in Brain Regions of a Triple Transgenic Model of Alzheimer's Disease

Abstract

A disruption to circadian rhythmicity and the sleep/wake cycle constitutes a major feature of Alzheimer's disease (AD). The maintenance of circadian rhythmicity is regulated by endogenous clock genes and a number of external Zeitgebers, including light. This study investigated the light induced changes in the expression of clock genes in a triple transgenic model of AD (3×Tg-AD) and their wild type littermates (Non-Tg). Changes in gene expression were evaluated in four brain areas¾suprachiasmatic nucleus (SCN), hippocampus, frontal cortex and brainstem¾of 6- and 18-month-old Non-Tg and 3×Tg-AD mice after 12 h exposure to light or darkness. Light exposure exerted significant effects on clock gene expression in the SCN, the site of the major circadian pacemaker. These patterns of expression were disrupted in 3×Tg-AD and in 18-month-old compared with 6-month-old Non-Tg mice. In other brain areas, age rather than genotype affected gene expression; the effect of genotype was observed on hippocampal Sirt1 expression, while it modified the expression of genes regulating the negative feedback loop as well as Rorα, Csnk1ɛ and Sirt1 in the brainstem. In conclusion, during the early development of AD, there is a disruption to the normal expression of genes regulating circadian function after exposure to light, particularly in the SCN but also in extra-hypothalamic brain areas supporting circadian regulation, suggesting a severe impairment of functioning of the clock gene pathway. Even though this study did not demonstrate a direct association between these alterations in clock gene expression among brain areas with the cognitive impairments and chrono-disruption that characterize the early onset of AD, our novel results encourage further investigation aimed at testing this hypothesis.

Keywords: Aging; Alzheimer’s disease; clock genes; light exposure; suprachiasmatic nucleus.

Fig.1

Clock gene expression level in the suprachiasmatic nucleus (SCN) of 6-month-old and 18-month-old Non-Tg and 3×Tg-AD mice after 12 h exposure to darkness (D) or light (L). Data are expressed as mean±SEM of fold change values and were analyzed by 3-way ANOVA. Tukey’s post-hoc test was used where appropriate to perform multiple comparisons. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Fig.2

Clock gene expression level in the hippocampus (H) of 6-month-old and 18-month-old Non-Tg and 3×Tg-AD mice after 12 h exposure to darkness (D) or light (L). Data are expressed as mean±SEM of fold change values and analyzed by 3-way ANOVA. Tukey’s post hoc test was used where appropriate to perform multiple comparisons. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Fig.3

Clock gene expression level in the frontal cortex (FC) of 6-month-old and 18-month-old Non-Tg and 3×Tg-AD mice after 12 h exposure to darkness (D) or light (L). Data are expressed as mean±SEM of fold change values and analyzed by 3-way ANOVA. Tukey’s post hoc test was used where appropriate to perform multiple comparisons. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Fig.4

Clock gene expression level in the brainstem (BS) of 6-month-old and 18-month-old Non-Tg and 3×Tg-AD mice after 12 h exposure to darkness (D) or light (L). Data are expressed as mean±SEM of fold change values and analyzed by 3-way ANOVA. Tukey’s post hoc test was used where appropriate to perform multiple comparisons. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Fig.5

a) Representative microphotographs (10×magnification, scale bar 100 μm) and results obtained from the semi-quantitative analyses of Aβ (6E10 antibody) and tau (HT7 antibody) immunostaining from Non-Tg (n = 3, white bars) and 3×Tg-AD (n = 3, black bars) mice. The red squares within the brain diagrams illustrate the sites where the representative microphotographs were taken. The data are mean±SEM ^**p < 0.01 and ^***p < 0.001 versus age-matched Non-Tg mice (Unpaired Student’s t-test, n = 3). b-d) Scatterplot of Aβ or tau protein levels versus Per3 and Rorα mRNA expression showing a direct correlation (Pearson test) in the hippocampus of 3×Tg-AD mice.