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Expression of the human PAC1 receptor leads to dose-dependent hydrocephalus-related abnormalities in mice
Bing Lang, … , Anthony J. Harmar, Sanbing Shen
Bing Lang, … , Anthony J. Harmar, Sanbing Shen
Published July 3, 2006
Citation Information: J Clin Invest. 2006;116(7):1924-1934. https://doi.org/10.1172/JCI27597.
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Research Article Neuroscience

Expression of the human PAC1 receptor leads to dose-dependent hydrocephalus-related abnormalities in mice

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Abstract

Hydrocephalus is a common and potentially devastating birth defect affecting the CNS, and its relationship with G protein–coupled receptors (GPCRs) is unknown. We have expressed 2, 4, or 6 copies of a GPCR — the human PAC1 receptor with a 130-kb transgene in the mouse nervous system in a pattern closely resembling that of the endogenous gene. Consistent with PAC1 actions, PKA and PKC activity were elevated in the brains of Tg mice. Remarkably, Tg mice developed dose-dependent hydrocephalus-like characteristics, including enlarged third and lateral ventricles and reduced cerebral cortex, corpus callosum, and subcommissural organ (SCO). Neuronal proliferation and apoptosis were implicated in hydrocephalus, and we observed significantly reduced neuronal proliferation and massively increased neuronal apoptosis in the developing cortex and SCO of Tg embryos, while neurite outgrowth and neuronal migration in vitro remain uncompromised. Ventricular ependymal cilia are crucial for directing cerebrospinal fluid flow, and ependyma of Tg mice exhibited disrupted cilia with increased phospho-CREB immunoreactivity. These data demonstrate that altered neuronal proliferation/apoptosis and disrupted ependymal cilia are the main factors contributing to hydrocephalus in PAC1-overexpressing mice. This is the first report to our knowledge demonstrating that misregulation of GPCRs can be involved in hydrocephalus-related neurodevelopmental disorders.

Authors

Bing Lang, Bing Song, Wendy Davidson, Alastair MacKenzie, Norman Smith, Colin D. McCaig, Anthony J. Harmar, Sanbing Shen

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Figure 1

Genomic organization and expression of the human ADCYAP1R1 transgene.

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Genomic organization and expression of the human ADCYAP1R1 transgene.
(A...
(A) Mapping of the human ADCYAP1R1 gene encoding the PAC1 receptor to 7p between STS markers sWss1736 and D7S2679. (B) Overlapping of 221D1 with 204D22 at the 3′ half of the PAC1 receptor coding region. (C) Determination of transgene copy number by semiquantitative PCR with primers PAC1For and PAC1Rev. The ratios of the 1.8-kb band (human) to the 1.4-kb fragment (mouse) indicated that Tg1 (lane 1), Tg2 (lane 2), and Tg3 (lane 3) contained 6, 4, and 2 copies of the transgene, respectively. (D) The expression of the human PAC1 mRNA in E10.5 mouse embryos detected by RT-PCR with the primers PAC1For and PAC1Rev. RsaI restriction digestion of the PCR products resulted in 314-bp and 232-bp bands from human and mouse PAC1 mRNA, respectively. Human fetal cDNA (h) was included as a control. m, mouse. (E–G) X-gal staining of E10.5 mouse embryos derived from lines Tg1 (E), Tg2 (F), and Tg3 (G), revealing transgene expression in the nervous system. (H–M) X-gal staining of coronal sections of adult brains, showing high levels of transgene expression in anterodorsal thalamic nucleus (AD; J), anterior hypothalamic nucleus (AHA; L), amygdala (Amg; M), anterior olfactory nucleus (Ao; H), anteroventral thalamic nucleus (AV; J), cingulate cortex (Cg; I), dentate gyrus (DG; K and M), frontal cortex (Fr; I), piriform cortex (Pir; J), paraventricular nucleus of the thalamus (Pv; J), reuniens thalamic nucleus (Re; J), and ventricular ependymal layer (vel; I, K and M).
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