Pharmacological imposition of sleep slows cognitive decline and reverses dysregulation of circadian gene expression in a transgenic mouse model of Huntington's disease

Abstract

Transgenic R6/2 mice carrying the Huntington's disease (HD) mutation show disrupted circadian rhythms that worsen as the disease progresses. By 15 weeks of age, their abnormal circadian behavior mirrors that seen in HD patients and is accompanied by dysregulated clock gene expression in the circadian pacemaker, the suprachiasmatic nucleus (SCN). We found, however, that the electrophysiological output of the SCN assayed in vitro was normal. Furthermore, the endogenous rhythm of circadian gene expression, monitored in vitro by luciferase imaging of organotypical SCN slices removed from mice with disintegrated behavioral rhythms, was also normal. We concluded that abnormal behavioral and molecular circadian rhythms observed in R6/2 mice in vivo arise from dysfunction of brain circuitry afferent to the SCN, rather than from a primary deficiency within the pacemaker itself. Because circadian sleep disruption is deleterious to cognitive function, and cognitive decline is pronounced in R6/2 mice, we tested whether circadian and cognitive disturbances could be reversed by using a sedative drug to impose a daily cycle of sleep in R6/2 mice. Daily treatment with Alprazolam reversed the dysregulated expression of Per2 and also Prok2, an output factor of the SCN that controls behavioral rhythms. It also markedly improved cognitive performance of R6/2 mice in a two-choice visual discrimination task. Together, our data show for the first time that treatments aimed at restoring circadian rhythms may not only slow the cognitive decline that is such a devastating feature of HD but may also improve other circadian gene-regulated functions that are impaired in this disease.

Figure 1.

Disrupted activity–rest cycles and circadian expression of mPER1 and mPER2 in SCN of R6/2 mice. A, Representative actograms of WT (left) and R6/2 (right) mice held under 12 h light/dark cycle from 8 weeks of age onwards. Note progressive deterioration of activity profile in R6/2 mice. B, Group data (mean ± SEM) for interdaily stability (left) and intradaily variability (right) of WT (open circles) and R6/2 (filled circles) mice reveal significant deterioration from 11 weeks of age. C, Representative photomicrographs of SCN immunostained for mPer1 and mPer2 from WT and R6/2 mice samples at CT12 (projected lights off) and CT14. Note the early decline in mPER-immunoreactive (IR) in R6/2 mice. Scale bar, 500 μm. D, Group data (mean ± SEM; n > 3 per group) showing circadian rhythm of abundance of mPER1- and mPER2-IR nuclei in SCN of WT (open bars) and R6/2 (filled bars) mice. Note the early decline in mPER-IR in mutant SCN.

Figure 2.

R6/2 mutation disrupts activity–rest cycles in mPer1::Luciferase reporter mice used to analyze molecular time keeping in SCN in vitro. A, Representative actograms of WT (top) and R6/2 (bottom) mice held under 12 h light/dark cycle from 12 weeks of age onwards. Note the progressive deterioration of activity profile in R6/2 mice. B, Periodogram analyses of mice with circadian behavior that is illustrated in A. The dotted line denotes significant periodicity at p < 0.01. Note the highly significant peak in the WT but not in R6/2 mice. C, Mean daily activity profile of mice with circadian behavior that is illustrated in A, and corresponding ratio of activity in light phase to total activity, indicating more daytime and less nocturnal activity. Circadian bioluminescence recordings from SCN explants obtained from these two mice are depicted in Figure 3, A and B.

Figure 3.

Functional molecular time keeping in SCN of R6/2 and WT mice revealed in vitro by bioluminescence gene expression. A, B, Representative recordings of bioluminescence gene expression from SCN of WT (A) and R6/2 mice (B) with circadian behavior that is illustrated in Figure 2 A. Although there is a trend toward lower amplitude of circadian gene expression in R6/2 SCN, group data (mean ± SEM) reveal no significant difference in the in vitro period of WT (n = 7) and R6/2 (n = 6) SCN. Circadian cycles of gene expression in R6/2 mice are significantly advanced relative to WT controls, such that time to first peak is ∼1 h earlier. This relative phase difference between genotypic groups was maintained throughout the recording, because periods were equivalent. When expressed relative to initial peak amplitude (100%), the rate of dampening of circadian gene expression is not affected by the R6/2 mutation (C) (i.e., molecular time keeping is sustained equally in WT and R6/2 SCN) (mean ± SEM).

Figure 4.

The effect of Alprazolam and chloral hydrate on sleep and on rousability in R6/2 and WT mice. A, Alprazolam (black symbols) had a hypnotic effect on both R6/2 (circles) and WT (squares) mice that was pronounced at 1 h and had worn off by 4 h. C, Chronic treatment with Alprazolam from 4 weeks of age improved predosing rousability in R6/2 mice. This effect lasted for several weeks (D; data shown for 60 s after cage opening). R6/2 mice treated with chloral hydrate (black symbols) showed residual sedation, both before (data not shown) and 4 h after drug injection (B; data shown for 60 s after cage opening). *p < 0.05, **p < 0.01, and ***p < 0.001: statistical significance of Alprazolam- or chloral hydrate-treated R6/2 mice compared with vehicle-treated R6/2 mice.

Figure 5.

The effect of Alprazolam and chloral hydrate treatments on performance in the two-choice swim tank task. Alprazolam and chloral hydrate treatments (black symbols) had no effect on the performance of WT mice (squares; A, C). In R6/2 mice (circles), Alprazolam treatment started either from 4 weeks (A) or 9 weeks (B) of age markedly improved acquisition learning in the two-choice swim tank at 12–13 weeks of age. C, R6/2 mice treated with chloral hydrate performed better than vehicle-treated mice during both acquisition and reversal of the two-choice swim tank task. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6.

The effect of Alprazolam treatment on motor function and survival. A, Alprazolam treatment (black symbols) did not affect rotarod performance of the WT mice (squares). Alprazolam treatment, started from 4 weeks of age, improved motor abilities of R6/2 mice (circles) on the rotarod at only one speed, 15 rpm (A). These mice did not survive longer than the vehicle-treated R6/2 mice (B). C, Alprazolam treatment, started from 10 weeks of age, improved motor abilities of R6/2 mice on the rotarod at all speeds. These mice showed a significant improvement in early survival (first 50% of mice to die) compared with the vehicle-treated R6/2 mice (D). *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7.

The effect of Alprazolam treatment on mRNA expression of clock genes in the SCN. Expression profiles are shown of mBmal1 (A, B), mPer2 (C, D), and mProk2 (E, F) from vehicle-treated (A, C, E) and Alprazolam-treated (B, D, F) mice. Significant differences were seen in expression levels of these genes in the SCN of vehicle-treated R6/2 mice (open circles) compared with vehicle-treated WT mice (open squares). In R6/2 mice, Alprazolam reversed the dysregulated gene expression of mPer2 (D) and mProk2 (F).