Cry1Δ11 mutation induces ADHD-like symptoms through hyperactive dopamine D1 receptor signaling
Dengfeng Liu, Zhengyu Xie, Panyang Gu, Xiangyu Li, Yichun Zhang, Xinying Wang, Zhiheng Chen, Suixin Deng, Yousheng Shu, Jia-Da Li
Dengfeng Liu, Zhengyu Xie, Panyang Gu, Xiangyu Li, Yichun Zhang, Xinying Wang, Zhiheng Chen, Suixin Deng, Yousheng Shu, Jia-Da Li
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Research Article Neuroscience

Cry1Δ11 mutation induces ADHD-like symptoms through hyperactive dopamine D1 receptor signaling

Abstract

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder that affects approximately 5.3% of children and approximately 2.5% of adults. There is an intimate relationship between ADHD and sleep disturbance. Specifically, individuals carry a mutation in the core circadian gene CRY1 (c. 1657 + 3A > C), which results in the deletion of exon 11 expression in the CRY1 protein (CRY1Δ11), causing them to exhibit typical ADHD symptoms. However, the underlying mechanism is still elusive. In this study, we demonstrate that Cry1Δ11 (c. 1717 + 3A > C) mice showed ADHD-like symptoms, including hyperactivity, impulsivity, and deficits in learning and memory. A hyperactive cAMP signaling pathway was found in the nucleus accumbens (NAc) of Cry1Δ11 mice. We further demonstrated that upregulated c-Fos was mainly localized in dopamine D1 receptor-expressing medium spiny neurons (DRD1-MSNs) in the NAc. Neuronal excitability of DRD1-MSNs in the NAc of Cry1Δ11 mice was significantly higher than that of WT controls. Mechanistically, the CRY1Δ11 protein, in contrast to the WT CRY1 protein, failed to interact with the Gαs protein and inhibit DRD1 signaling. Finally, the DRD1 antagonist SCH23390 normalized most ADHD-like symptoms in Cry1Δ11 mice. Thus, our results reveal hyperactive DRD1 signaling as an underlying mechanism and therapeutic target for ADHD induced by the highly prevalent CRY1Δ11 mutation.

Authors

Dengfeng Liu, Zhengyu Xie, Panyang Gu, Xiangyu Li, Yichun Zhang, Xinying Wang, Zhiheng Chen, Suixin Deng, Yousheng Shu, Jia-Da Li

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Figure 1

Generation of Cry1Δ11-knockin mice.

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Generation of Cry1Δ11-knockin mice. (A) Schematic representation of mous...
(A) Schematic representation of mouse Cry1 protein (top), genomic structure (middle), and aa sequence encoded by exon 11 of human or mouse Cry1 gene (bottom). (B) Comparison of the nucleotide sequences at the junction of exon 11 and intron 11 of human and mouse Cry1 genes. The mutation (A > C) is labeled in red; the 96-bp sequences of donor DNA and sgRNA used for gene editing are shown at the bottom. (C) Sanger sequencing of the junction between exon 11 and intron 11 confirmed the existence of the desired nucleotide modification (c. 1717 + 3A > C). Mouse tail genomic DNA was amplified by PCR and the product was analyzed by Sanger sequencing. (D) Sanger sequencing of the cDNA confirmed deletion of exon 11 in the Cry1 (c. 1717 + 3A > C) mutant mouse. RNA samples extracted from the livers of WT and mutant mice were reverse transcribed to cDNA. The cDNA was amplified by PCR and the products from mutant mice were cloned into a pMD20-T vector by TA cloning (Takara, 6028). Sanger sequencing showed that the top band corresponded to the WT Cry1, whereas the bottom band lacked exon 11. (E) Western blot confirmed that the c. 1717 + 3A > C mutation resulted in a truncated Cry1 protein. Proteins from the livers of WT and mutant mice were subjected to Western blot with a Cry1 Ab. (F) Representative double-plotted actograms of WT and Cry1Δ11 mice. (G) The free-running periods of WT (n = 8) and Cry1Δ11 mice (n = 6); P > 0.05, unpaired Student’s t test. Data are presented as mean ± SEM.