LOSS OF AKAIN1, AN ENDOGENOUS MICROPROTEIN THAT INHIBITS PKA LOCALIZATION, ATTENUATES CONTEXTUAL DISCRIMINATION AND BEHAVIORAL FLEXIBILITY IN MICE
Authors: Fujii Kazuki , Koshidaka Yumie , Adachi Mayumi , Yanagibashi Yuko , Matsuo Mina , Hongo Shoko , Nishizono Hirofumi , Takasaki Ichiro , Kurosawa Nobuyuki , Aizawa Yasunori , Takao *Keizo 
Published: February 2025

Abstract

Background
By definition, long noncoding RNAs (lncRNAs) contain no protein-coding open reading frames (ORFs). Recent bioinformatics and high-throughput sequencing studies, however, demonstrated that many lncRNAs possess short “non-canonical” ORFs (sORFs) encoding microprotein. These newly identified microprotein may help to elucidate the mechanisms underlying vital phenomena and are potential targets for drug discovery. A-kinase anchor inhibitor 1 (Akain1) is a protein kinase A (PKA)-binding microprotein encoded by an sORF. Specific PKA actions are achieved by controlling its cellular localization through a family of A-kinase anchoring proteins (AKAPs). AKAPs localize PKA to specific intracellular sites and spatially restrict intracellular signaling events. In PKA signaling, Akain1 competes with other AKAPs (e.g., AKAP1 and MAP2) to bind PKA and seems to cancel the intracellular localization of PKA. AKAP-PKA interactions control various cellular processes, including the regulation of neuroplasticity, and Akain1 mRNA is preferentially expressed in neural tissues. Whether Akain1 is translated into an endogenous protein in vivo and how Akain1 affects brain function and behavior, however, remain unclear.

Aims & Objectives
We aimed to elucidate the existence and function of Akain1 microprotein in vivo.

Method
We generated Akain1 reporter mice and Akain1 knockout (KO) mice on the C57BL/6J background using CRISPR-Cas9 genome editing system. To investigate the function of Akain1 in the brain, we subjected Akain1 KO mice to the comprehensive behavioral test battery.

Results
Utilizing Akain1 reporter mice in which an epitope-tag coding sequence was inserted before the stop codon, we detected epitope-tag-specific cells in the mouse central nervous system (e.g., cortex, superior colliculus, cerebellum, etc.). These findings suggest that Akain1 is translated into an endogenous protein.Akain1 KO mice demonstrated some behavioral phenotypes in the comprehensive behavioral test battery. In the pattern separation test, Akain1 KO mice exhibited impaired performance in distinguishing between two similar contexts. In the Barnes maze test, Akain1 KO mice and wild-type (WT) mice learned the fixed escape box position at a comparable rate. In reversal learning, in which the target was moved to the opposite side of the maze, Akain1 KO mice and WT mice learned the new escape box position at a similar rate. While WT mice spent significantly more time investigating the new target than the original target, Akain1 KO mice exhibited no significant difference, suggesting that Akain1 KO mice had behavioral flexibility deficits.

Discussion & Conclusion
Together, these findings indicate that Akain1 is translated in the brain and has a critical role in the discrimination of similar environmental contexts. Further investigation is necessary to elucidate the molecular mechanism of how Akain1 regulates these behavioral phenotypes. Nonetheless, our results underscore the significance of the microprotein Akain1 in the central nerve system.