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Identification of novel mouse and rat CB1R isoforms and in silico modeling of human CB1R for peripheral cannabinoid therapeutics

Acta Pharmacologica Sinica (2018) | Download Citation

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Abstract

Targeting peripheral CB1R is desirable for the treatment of metabolic syndromes without adverse neuropsychiatric effects. We previously reported a human hCB1b isoform that is selectively enriched in pancreatic beta-cells and hepatocytes, providing a potential peripheral therapeutic hCB1R target. It is unknown whether there are peripherally enriched mouse and rat CB1R (mCB1 and rCB1, respectively) isoforms. In this study, we found no evidence of peripherally enriched rodent CB1 isoforms; however, some mCB1R isoforms are absent in peripheral tissues. We show that the mouse Cnr1 gene contains six exons that are transcribed from a single promoter. We found that mCB1A is a spliced variant of extended exon 1 and protein-coding exon 6; mCB1B is a novel spliced variant containing unspliced exon 1, intron 1, and exon 2, which is then spliced to exon 6; and mCB1C is a spliced variant including all 6 exons. Using RNAscope in situ hybridization, we show that the isoforms mCB1A and mCB1B are expressed at a cellular level and colocalized in GABAergic neurons in the hippocampus and cortex. RT-qPCR reveals that mCB1A and mCB1B are enriched in the brain, while mCB1B is not expressed in the pancreas or the liver. Rat rCB1R isoforms are differentially expressed in primary cultured neurons, astrocytes, and microglia. We also investigated modulation of Cnr1 expression by insulin in vivo and carried out in silico modeling of CB1R with JD5037, a peripherally restricted CB1R inverse agonist, using the published crystal structure of hCB1R. The results provide models for future CB1R peripheral targeting.

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Acknowledgements

JME, Q-RL, NSH, IG-M, JFO, and SS-C-C are supported by the Intramural Research Program of the National Institute on Aging; Z-XX by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health; HQ by the National Science Foundation of China (31671375); ESO by NIH grant DA032890; and YW by the National Health Research Institutes and a Central Government S & T Grant, Taiwan (106-1901-01-10-02).

Author contributions

Q-RL, ESO, and JME conceptualized the study and wrote the manuscript. Q-RL, JME, JFO, IG-M, MD, and SS-C-C performed S961 treatment and mouse and rat tissue dissections. Q-RL and HQ analyzed the gene structures and protein modeling, respectively, using the bioinformatics tools. Q-RL, NSH, and Z-XX performed RT-qPCR, RNAscope ISH, imaging, and data analysis. Primary cell culture and isolation of neurons, astrocytes, and microglia were conducted by YW. All authors reviewed and approved the publication of this manuscript.

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Affiliations

  1. Laboratory of Clinical Investigation, National Institute on Aging, NIH, Baltimore, MD, USA

    • Qing-Rong Liu
    • , Nicholas S. Huang
    • , Jennifer F. O’Connell
    • , Isabel Gonzalez-Mariscal
    • , Sara Santa-Cruz-Calvo
    • , Maire E. Doyle
    •  & Josephine M. Egan
  2. Center for Bioinformatics, State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China

    • Hong Qu
  3. Addiction Biology Unit, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, NIH, Baltimore, MD, USA

    • Zheng-Xiong. Xi
  4. Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan

    • Yun Wang
  5. Department of Biology, William Paterson University, Wayne, NJ, USA

    • Emmanuel. S. Onaivi

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The authors declare no competing interests.

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Correspondence to Qing-Rong Liu.

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https://doi.org/10.1038/s41401-018-0152-1