Ultanir lab

Kinases and Brain Development Laboratory

: CDKL5

CDKL5’s role in neuronal microtubules

Loss-of-function mutations in the X-linked CDKL5 gene cause a severe neurodevelopmental disorder with epilepsy and profound sensory, motor and learning defects. CDKL5 deficiency disorder (CDD) is a rare disorder with no disease-targeting therapies. We set out to determine CDKL5 direct substrates using our expertise in chemical genetics and proteomics. We identified the first physiological substrates of CDKL5 to be microtubule binding proteins EB2, MAP1S and ARHGEF2 (Baltussen et al, 2018, EMBO Journal).

EB2 phosphorylation, has since been used as a successful preclinical biomarker successfully used in numerous academic and industry labs to measure CDKL5’s activity. We were given “Lab of the year 2018” award by the Loulou Foundation dedicated to advancing CDKL5 research for our contributions. We have confirmed the novel substrates in humans using iPSC derived neurons.

More recently, using a novel CRISPR engineered MAP1S phosphomutant mouse model, and live-imaging in primary cultures, we showed that CDKL5 is necessary for dynein-mediated transport in dendrites during neuronal development, which critically is required for learning and memory (Lopes et al, in preparation).  

CDKL5’s role in regulating ion channels

Through our rigorous use of proteomics and mass spectrometry, we identified multiple CDKL5’s substrates and began to comprehensively reveal its multiple functions. Recently, we discovered that CDKL5 phosphorylates voltage gated Ca2+ channel Cav2.3 subunit CACNA1E and regulates neuronal excitability (Sampedro-Castaneda et al 2023, Nature Communications). 

CDKL5’s phosphorylation of Cav2.3 suppresses its activity, consequently loss-of this phosphorylation leads to increased calcium influx. Electrophysiological recordings from HEK293 cells expressing recombinant Cav2.3 and recordings from acute slices from Cav2.3 phosphomutant mouse model indicate that Cav2.3 has increased decay kinetics and enhanced response to GPCR mediated activation in the absence of phospho- Ser15.

Interestingly, point mutations in CACNA1E cause gain-of-function phenotypes in patients with a developmental and epileptic encephalopathy, similar to CDD, indicating that Cav2.3 can be a critical substrate mediating CDKL5 pathology in epilepsy. Our recent discoveries point towards additional ion channel components being phosphorylated by CDKL5.

How one kinase orchestrates these seemingly diverse functions is an open question, and we aim to study regulation and local activation of CDKL5 in neurons.

CDKL5’s roles in gene regulation 

CDKL5 is expressed in most, if not all, neuronal subtypes. Its expression is increased during late embryonic and early postnatal development. CDKL5 substrate phosphorylations are at their highest levels in early postnatal stages, implicating CDKL5 in early brain development.

Results from our lab and others have revealed that CDKL5 phosphorylates nuclear substrates and contribute to gene regulation. How CDKL5 regulates differentiation in different neuronal cell types by affecting transcription is not well-understood. Some of our research focus on CDKL5’s roles in gene regulation.

Interestingly, CDKL5 levels persist in adulthood, while substrate phosphorylations are reduced. We hypothesize that CDKL5 regulates synaptic plasticity throughout adulthood, we aim to pursue this using cellular as well as circuit level learning paradigms. Recent collaborative work in developing specific CDKL5 inhibitor compounds (Castano et al, 2023, eLife) will allow us to acutely inhibit CDKL5 and test its effects in gene expression. 

Translating our findings in future therapies for CDD

Targeting Cav2.3:  In collaboration with Lario Therapeutics, we are seeking to test if novel small-molecule inhibitors of Cav2.3 can reduce increased excitability observed in CDKL5 knockout iPSC derived neurons. Alternatively, we are testing seizure susceptibility paradigms in CDKL5 knockout mice and are testing if inhibitors can reduce seizures. 

CDKL2, as a target for CDD: We have recently identified that a paralog of CDKL5, CDKL2, can phosphorylate CDKL5 targets in the brain (Silvestre et al, 2024, Molecular Psychiatry). The discovery of CDKL2 brings the possibility of increasing CDKL2 levels by targeting its transcription or translation using small molecule or alternative modalities.

Due to being X-linked and X chromosome inactivation, increasing CDKL5 levels is not a prominent path to therapy, making CDKL2 a significant discovery. Owing to our translational findings, we received our second “Lab of the year” award from Loulou foundation in 2023.

Press release: Researchers identify new way to treat genetic epilepsy by replacing ‘lost’ enzymev