Cnpy32xHA mice reveal neuronal expression of Cnpy3 in the brain

Identification of biallelic CNPY3 mutations in patients with epileptic encephalopathy and abnormal electroencephalography findings of Cnpy3 knock-out mice have indicated that the loss of CNPY3 function causes neurological disorders such as epilepsy. However, the basic property of CNPY3 in the brain...

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Published in:Journal of neuroscience methods 2023-01, Vol.383, p.109730-109730, Article 109730
Main Authors: Islam, Md. Monirul, Mutoh, Hiroki, Aoto, Kazushi, Belal, Hazrat, Saitsu, Hirotomo
Format: Article
Language:English
Subjects:
Cnpy3
HA epitope tag
i-GONAD
Neuronal expression
Protein degradation
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Summary:Identification of biallelic CNPY3 mutations in patients with epileptic encephalopathy and abnormal electroencephalography findings of Cnpy3 knock-out mice have indicated that the loss of CNPY3 function causes neurological disorders such as epilepsy. However, the basic property of CNPY3 in the brain remains unclear. We generated C-terminal 2xHA-tag knock-in Cnpy3 mice by i-GONAD in vivo genome editing system to investigate the expression and function of Cnpy3 in the mouse brain. 2xHA-tagged Cnpy3 was confirmed by immunoblot analysis using anti-HA and CNPY3 antibodies, although HA tagging caused the decreased Cnpy3 protein level. Immunohistochemical analysis of Cnpy32xHA knock-in mice showed that Cnpy3-2xHA was predominantly expressed in the neuron. In addition, Cnpy3 and Cnpy3-2xHA were both localized in the endoplasmic reticulum and synaptosome and showed age-dependent expression changes in the brain. Conventional Cnpy3 antibodies could not allow us to investigate the distribution of Cnpy3 expression in the brain, while HA-tagging revealed the expression of CNPY3 in neuronal cells. Taken together, we demonstrated that Cnpy32xHA knock-in mice would be useful to further elucidate the property of Cnpy3 in brain function and neurological disorders. •The generation of mice with HA-tagged Cnpy3 revealed that the protein is predominantly expressed in the neuron.•Cnpy3 was localized not only in the endoplasmic reticulum but also synaptosome.•Cnpy3 was highly expressed during the early postnatal period in the brain.•C terminus HA-tagging accelerated the degradation of the Cnpy3 protein.
ISSN:0165-0270
1872-678X
DOI:10.1016/j.jneumeth.2022.109730