Abstract

CDKL5 is an X-linked serine/threonine kinase predominantly expressed in the mammalian brain. Loss-of-function mutations in CDKL5 lead to CDKL5 Deficiency Disorder (CDD), a rare neurodevelopmental disease characterized by early-onset seizures, profound developmental impairments and a lifelong need for care. To investigate CDKL5 ’s role in brain development, we utilized chemical genetic and proteomics approaches to identify its substrates in the mouse brain. This research uncovered several physiological substrates, including microtubule-binding proteins (EB2, MAP1S , and ARHGEF2 ) and the voltage-gated calcium channel Cav2.3. To explore the functional significance of these phosphorylations, we developed phosphomutant mouse models for MAP1S and Cav2.3. Our findings indicate that MAP1S phosphorylation influences microtubule binding, stabilizes dendritic microtubules, regulates tubulin tyrosination, and supports dynein-mediated transport in neuronal dendrites. Disruptions in these processes lead to impaired synapse development and learning deficits. Similarly, our studies on Cav2.3 reveal that loss of its phosphorylation results in a gain-of-function effect, mimicking the impact of ultrarare CACNA1E mutations found in epilepsy patients. These results suggest that inhibiting Cav2.3 activity could provide therapeutic benefits for CDD . We also discovered that CDKL2 phosphorylates several CDKL5 substrates in the brain, raising the possibility that enhancing CDKL2 or related kinases could offer a therapeutic strategy for CDD .