Förster resonance energy transfer-based kinase mutation phenotyping reveals an aberrant facilitation of Ca2+/calmodulin-dependent CaMKIIα activity in de novo mutations related to intellectual disability

Hajime Fujii; Hiroyuki Kidokoro; Yayoi Kondo; Masahiro Kawaguchi; Shin-Ichiro Horigane; Jun Natsume; Sayaka Takemoto-Kimura; Haruhiko Bito

doi:10.3389/fnmol.2022.970031

Förster resonance energy transfer-based kinase mutation phenotyping reveals an aberrant facilitation of Ca²⁺/calmodulin-dependent CaMKIIα activity in de novo mutations related to intellectual disability

Front Mol Neurosci. 2022 Sep 1:15:970031. doi: 10.3389/fnmol.2022.970031. eCollection 2022.

Authors

Hajime Fujii¹, Hiroyuki Kidokoro², Yayoi Kondo¹, Masahiro Kawaguchi², Shin-Ichiro Horigane^{3

4}, Jun Natsume^{2

5}, Sayaka Takemoto-Kimura^{3

4}, Haruhiko Bito¹

Affiliations

¹ Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
² Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
³ Department of Neuroscience I, Research Institute of Environmental Medicine (RIEM), Nagoya University, Nagoya, Japan.
⁴ Department of Molecular/Cellular Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan.
⁵ Department of Developmental Disability Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Abstract

CaMKIIα plays a fundamental role in learning and memory and is a key determinant of synaptic plasticity. Its kinase activity is regulated by the binding of Ca²⁺/CaM and by autophosphorylation that operates in an activity-dependent manner. Though many mutations in CAMK2A were linked to a variety of neurological disorders, the multiplicity of its functional substrates renders the systematic molecular phenotyping challenging. In this study, we report a new case of CAMK2A P212L, a recurrent mutation, in a patient with an intellectual disability. To quantify the effect of this mutation, we developed a FRET-based kinase phenotyping strategy and measured aberrance in Ca²⁺/CaM-dependent activation dynamics in vitro and in synaptically connected neurons. CaMKIIα P212L revealed a significantly facilitated Ca²⁺/CaM-dependent activation in vitro. Consistently, this mutant showed faster activation and more delayed inactivation in neurons. More prolonged kinase activation was also accompanied by a leftward shift in the CaMKIIα input frequency tuning curve. In keeping with this, molecular phenotyping of other reported CAMK2A de novo mutations linked to intellectual disability revealed aberrant facilitation of Ca²⁺/CaM-dependent activation of CaMKIIα in most cases. Finally, the pharmacological reversal of CAMK2A P212L phenotype in neurons was demonstrated using an FDA-approved NMDA receptor antagonist memantine, providing a basis for targeted therapeutics in CAMK2A-linked intellectual disability. Taken together, FRET-based kinase mutation phenotyping sheds light on the biological impact of CAMK2A mutations and provides a selective, sensitive, quantitative, and scalable strategy for gaining novel insights into the molecular etiology of intellectual disability.

Keywords: CaMKII; FRET; de novo mutation; imaging; intellectual disability; neurodevelopmental disorders.