Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.

Lucas L Baltussen,Sila K Ultanir,Suzanne Claxton,Helen R Flynn,Alysson R Muotri,Ambrosius P Snijders,Margaux Silvestre,Priscilla D Negraes,Max Moeskops,Evangelos Christodoulou

doi:10.15252/embj.201899763

Abstract

Loss‐of‐function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early‐onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule‐associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3‐labelled plus‐end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient‐derived human neurons. Our results reveal a novel activity‐dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder.

Highlights

Loss-of-function mutations in Cyclin-dependent kinase-like 5 (CDKL5) kinase cause severe neurodevelopmental delay and early-onset seizures
We show that EB2 Ser222 and microtubule-associated protein 1S (MAP1S) Ser812 phosphorylations are dramatically reduced in the CDKL5 knockout mouse brain, making them bona fide phosphorylation sites for CDKL5
We found that ARHGEF2 Ser122 and MAP1S Ser786 phosphorylation levels were increased with CDKL5 overexpression, demonstrating that these substrates can be phosphorylated by CDKL5 in cells, while EB2 Ser222 and MAP1S Ser812 sites were already highly phosphorylated endogenously (Fig EV2A–C)

Summary

Introduction

Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. We found that CDKL5 phosphorylates three microtubuleassociated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder

Methods

Results

Conclusion