OVEREXPRESSION OF VASOACTIVE INTESTINAL PEPTIDE RECEPTOR 2 IN NEURONS CAUSES ABNORMAL DENDRITIC MORPHOLOGY IN THE PREFRONTAL CORTEX AND COGNITIVE IMPAIRMENT
Authors: Ono *Ami , Koan Daichi , Asano Satoshi , Miyaoka Tatsunori , Chen Lu , Nakagawa Shinsaku , Hayata-Takano Atsuko , Nakazawa Takanobu , Harada Akihiro , Hashimoto Hitoshi , Waschek James A , Tanimoto Kotaro , Ago Yukio 
Published: February 2025

Abstract

Background
Accumulating evidence indicates that a number of rare copy number variants have been strongly associated with schizophrenia and autism spectrum disorder (ASD). Clinical studies have shown that microduplications at 7q36.3, containing vasoactive intestinal peptide (VIP) receptor 2 (VIPR2), are a risk factor for schizophrenia and ASD (Vacic et al., 2011; Levinson et al., 2011; Yuan et al., 2014; Li et al., 2016; Marshall et al., 2017). VIPR2 encodes VPAC2, a seven transmembrane heterotrimeric G protein- coupled receptor that binds two homologous neuropeptides with high affinity, VIP and pituitary adenylate cyclase-activating polypeptide (Harmar et al., 2014). VIPR2 is widely distributed throughout the body in humans and is highly expressed in the peripheral and central nervous systems. In the brain, VIPR2 is expressed in neurons, astrocytes, and oligodendrocytes, but not at detectable levels in microglia. Mechanisms by which overactive VPAC2 receptor signaling may lead to these psychiatric disorders remain elusive.

Aims & Objectives
To investigate how increased VIPR2 dosage might predispose to psychiatric disorders, we aimed to determine the effects of neuron-specific overexpression of human VIPR2 in mice on dendritic morphology and behaviors.

Method
We generated a new line of transgenic mice that harbored a human VIPR2 (hVIPR2)-IRES-mCherry expression cassette placed downstream of a tetracycline-responsive element-regulated promoter (Ago et al., 2023). Mice thus generated were bred with ROSA:LNL:tTA mice, which express the tTA (Tet-off) in tissues undergoing Cre-mediated recombination. The ROSA:LNL:tTA-VIPR2-mCherry double transgenic mice were then bred with a tau-Cre mouse strain which has been reported target recombinase activity to neurons (Muramatsu et al., 2008).

Results
Immunohistochemical staining revealed the expression of mCherry, which is supposed to co-express with hVIPR2, throughout the brain, especially in the cerebral cortex, hippocampus, and amygdala, in hVIPR2 overexpressing mice. The co-localization of mCherry and a neuronal marker NeuN, but not an astrocyte marker GFAP or a microglia marker Iba1, was observed. We further confirmed by western blotting analysis that human VIPR2 is expressed in the prefrontal cortex of (PFC) of hVIPR2 overexpressing mice. Morphological analyses revealed reductions in cell body size, dendritic length, and branch number of PFC pyramidal neurons in those mice. hVIPR2 overexpressing mice also showed cognitive impairment in the novel object recognition test. To clarify the molecules and signaling pathways involved in the effects of neuron-specific VIPR2 overexpression, we performed RNA sequencing of PFC from hVIPR2 overexpressing mice. We identified 20 up-regulated and 18 down-regulated genes in hVIPR2 overexpressing mice, compared with littermates not harboring the tau-Cre transgene. Among the protein-coding genes, Rsc1a1 was the most up-regulated gene in mice overexpressing hVIPR2, and Lhx9 was the most down- regulated. Additionally, gene ontology and KEGG pathway enrichment analyses revealed that pathways regulating tyrosine metabolism, melanogenesis, and pluripotency of stem cells are activated in the PFC of hVIPR2 overexpressing mice.

Discussion & Conclusion
These results indicate that VIPR2 plays a crucial role in brain morphogenesis and suggest that overactive VIPR2 signaling during neuronal development plays a mechanistic role in some forms of schizophrenia and ASD.

References
Ago et al., Exp Neurol 362: 114339, 2023.
Harmar et al., Endocrinology 145:1203-1210, 2014.
Levinson et al., Am J Psychiatry 168: 302-316, 2011.
Li et al., Biol Psychiatry 80: 331-337, 2016.
Marshall et al., Nat Genet 49: 27-35, 2017.
Muramatsu et al, Biochem Biophys Res Commun 370: 419-423, 2008. Vacic et al., Nature 471: 499-503, 2011.
Yuan et al., Schizophr Res 156: 66-70, 2014.