Increased orexin expression promotes sleep/wake disturbances in the SOD1-G93A mouse model of amyotrophic lateral sclerosis

Abstract

Background: Sleep/wake disturbances in patients with amyotrophic lateral sclerosis (ALS) are well-documented, however, no animal or mechanistic studies on these disturbances exist. Orexin is a crucial neurotransmitter in promoting wakefulness in sleep/wake regulation, and may play an important role in sleep disturbances in ALS. In this study, we used SOD1-G93A transgenic mice as an ALS mouse model to investigate the sleep/wake disturbances and their possible mechanisms in ALS.

Methods: Electroencephalogram/electromyogram recordings were performed in SOD1-G93A transgenic mice and their littermate control mice at the ages of 90 and 120 days, and the samples obtained from these groups were subjected to quantitative reverse transcriptase-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay.

Results: For the first time in SOD1-G93A transgenic mice, we observed significantly increased wakefulness, reduced sleep time, and up-regulated orexins (prepro-orexin, orexin A and B) at both 90 and 120 days. Correlation analysis confirmed moderate to high correlations between sleep/wake time (total sleep time, wakefulness time, rapid eye movement [REM] sleep time, non-REM sleep time, and deep sleep time) and increase in orexins (prepro-orexin, orexin A and B).

Conclusion: Sleep/wake disturbances occur before disease onset in this ALS mouse model. Increased orexins may promote wakefulness and result in these disturbances before and after disease onset, thus making them potential therapeutic targets for amelioration of sleep disturbances in ALS. Further studies are required to elucidate the underlying mechanisms in the future.

Figure 1

Disturbances in sleep and wakefulness in SOD1-G93A transgenic mice. EEG/EMG recordings for 24 hours from the SOD1-G93A transgenic mice and littermate control mice at 90 days and 120 days of age. (a) TST, NREM, and DS are significantly decreased in the 90-day SOD1-G93A transgenic mice. WAKE is significantly increased. (b) In the 120-day SOD1-G93A transgenic mice, WAKE is also significantly enhanced. Besides TST, NREM, and DS, REM is also significantly reduced. Data are expressed as mean ± standard error (n = 6–8/group). *P < 0.05 and ^†P < 0.01. TST: Total sleep time; WAKE: Wakefulness; REM: Rapid eye movement sleep; NREM: Non-rapid eye movement sleep; LS: Light sleep; DS: Deep sleep; EEG/EMG: Electroencephalogram/electromyogram.

Figure 2

Prepro-orexin increases in SOD1-G93A transgenic mice. (a) Q-PCR was performed in the hypothalamic tissues from the SOD1-G93A transgenic mice and control groups. Prepro-orexin mRNA is significantly elevated in the hypothalamus of the 90 and 120 days SOD1-G93A transgenic mice, compared to control. (b) Western blotting was performed with antibody anti-orexin-prepro in the SOD1-G93A transgenic mice and control groups. Prepro-orexin expression is increased in the hypothalamus of the 90 and 120 days SOD1-G93A transgenic mice as compared to control. Data are expressed as mean ± standard error of mean (n = 4–6/group). *P < 0.05 and ^†P < 0.01. HT: hypothalamus; PCR: Polymerase chain reaction.

Figure 3

Orexin A and B increase in SOD1-G93A transgenic mice. (a-c) ELISA reveals that orexin A levels are enhanced in the hypothalamus and brain stem of the 90 and 120 days SOD1-G93A transgenic mice than controls, but there is no significant difference for orexin A in the CSF. (d-f) ELISA shows increased the level of orexin B in the hypothalamus and brain stem of the 90 and 120 days SOD1-G93A transgenic mice compared with control, but no significant difference is seen in the CSF. Data are expressed as mean ± standard error (n = 4–6/group). *P < 0.05 and ^†P < 0.01. HT: Hypothalamus; BS: Brain stem; CSF: Cerebrospinal fluid; ELISA: Enzyme-linked immunosorbent assay.

Figure 4

mRNA levels of orexin receptors. Q-PCR of orexin-1 receptor mRNA show no significant difference in the hypothalamus (a) and brain stem (b) of the 90 and 120 days SOD1-G93A transgenic mice than controls. No significant difference is found in orexin-2 receptor mRNA in the hypothalamus (c) and brain stem (d) of the 90 and 120 days SOD1-G93A transgenic mice than controls. HT: Hypothalamus; BS: Brain stem; PCR: Polymerase chain reaction.