Study on the Circadian Rhythm of Orexin System Genes and Their Impact on Early Alzheimer’s Disease (AD) Pathology

Research Background and Objectives

Alzheimer’s Disease (AD) is a chronic degenerative disease of the central nervous system, characterized by senile plaques formed by β-amyloid protein (Aβ) aggregation and neurofibrillary tangles formed by hyperphosphorylation of tau protein. Recent studies have found that Orexin and its receptors are closely related to the pathogenesis of AD, and under normal physiological conditions, Orexin system genes show circadian rhythms. However, the circadian characteristics of Orexin system genes in the early stages of AD and their potential role in AD progression remain unclear. This study aims to elucidate the circadian characteristics of Orexin system genes in the early stages of 3xtg-AD mice and explore their potential role in the development of AD neuropathology.

Paper Source and Authors

This article was written by researchers including Jing Yin, Chun-Mei Tuo, Kai-Yue Yu, Xiao-Hong Hu, Yan-Ying Fan, and Mei-Na Wu, all from Shanxi Medical University. The paper was published online in the journal “Neuromolecular Medicine” on October 16, 2023.

Research Process

a) Research Workflow

The study selected 6-month-old male 3xtg-AD mice and C57BL/6J (wild-type, WT) mice as experimental subjects, raised under constant 12-hour light-dark cycles (light from 6:00 AM to 6:00 PM) and constant temperature and humidity conditions. Samples were taken at ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20 according to Zeitgeber Time (ZT), i.e., light cycle time. QT-PCR method was used to detect the mRNA expression levels of Orexin system genes, AD risk genes, and core clock genes (ccgs) in the hypothalamus and hippocampus.

Simultaneously, Pearson correlation analysis was used to explore the correlation between the expression of Orexin system genes and AD risk genes or core clock genes, and Western Blot was used to detect the expression levels of soluble Aβ oligomers and phosphorylated tau (p-tau) protein in the hippocampus of 3xtg-AD mice.

b) Main Research Results

Circadian Characteristics of Orexin System Genes and AD Risk Genes in Hypothalamus and Hippocampus

1. Changes in the Hypothalamus:

   • In WT mice, the PPO gene showed circadian rhythmicity at ZT16; however, in 3xtg-AD mice, the PPO gene lost its circadian rhythmicity, and its expression level at ZT16 was significantly lower than in WT mice.
   • At ZT16, the expression levels of OX1R and OX2R genes in 3xtg-AD mice were significantly higher than in WT mice.
   • The expression of AD risk gene BACE2 at ZT16 was significantly higher than in WT mice, while BACE1 and BACE2 genes did not show significant circadian rhythms in all three types of mice.
   • Core clock genes Bmal1, Per1, Per2, and Cry1 showed obvious circadian rhythms in WT mice, but only the Per2 gene showed circadian rhythm in 3xtg-AD mice.

2. Changes in the Hippocampus:

   • In WT mice, OX1R and OX2R genes peaked at ZT16, while in 3xtg-AD mice, this peak was delayed to ZT20. The expression of OX1R (p = 0.003) and OX2R (p = 0.011) genes in WT mice at ZT16 was significantly higher than in 3xtg-AD mice.
   • The expression of AD risk gene Bace1 at ZT4 was significantly higher than in WT mice, but Bace2 and Bace1 genes did not show significant circadian rhythms in both types of mice.
   • Core clock genes Bmal1 and Cry2 showed significant circadian rhythms in WT mice but were insufficient in 3xtg-AD mice.

Expression of Soluble Aβ Oligomers and Phosphorylated Tau Protein

• The expression levels of soluble Aβ oligomers in 3xtg-AD mice at ZT6 and ZT18 were significantly higher than in WT mice but did not show circadian differences.
• The expression levels of p-tau in 3xtg-AD mice at ZT6 and ZT18 were significantly higher than in WT mice, and the expression at ZT18 was significantly higher than at ZT6.

Research Conclusions and Significance

Conclusions

The study preliminarily elucidated the circadian characteristics of Orexin system genes in the early stages of AD and confirmed the positive correlation between Orexin system genes and AD risk genes or core clock genes. Furthermore, the abnormal expression of these genes may accelerate the accumulation of Aβ and p-tau, thereby promoting the development of AD.

Research Value

This study reveals the potential role of the Orexin system in AD progression and further demonstrates the correlation between Orexin system genes and AD risk genes and clock genes. These findings have important scientific value for a comprehensive understanding of the pathological mechanisms of AD and may provide new insights for early intervention in AD.

Research Highlights

• First description of the circadian rhythm characteristics of Orexin system genes in 3xtg-AD mice and their relationship with AD pathology.
• Elucidation that abnormal expression of Orexin system genes in the early stages of AD may further affect AD progression by increasing the expression of Aβ and p-tau.
• The research methods employed various gene and protein expression level detection techniques, providing rich and empirically strong data.