New insight into CDKL5 deficiency disorder pathomechanism: phosphoproteomic profiling identifies SMAD3 as a novel downstream target of CDKL5

CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene, a member of a highly conserved family of serine-threonine kinases. Since kinase function alterations are associated with several pathologies, cancers, and also neurological disorders, these proteins can be both valuable biomarkers and potential drug targets for disease prognosis and treatment. Unfortunately, only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here we took advantage of a phospho-specific-antibody-microarray technology to identify potential direct CDKL5 substrates in a mouse model of the disorder. Among the potential CDKL5 targets, we show that SMAD3, a primary mediator of TGF-β action, is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF-β1 treatment normalized defective neuronal survival and maturation in Cdkl5 knockout (KO) neurons. Moreover, we demonstrate that Cdkl5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF-β1 prevents increased NMDA-induced cell death in hippocampal neurons from Cdkl5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of Cdkl5 KO mice. In conclusion, this study contributes to a better understanding of the molecular pathomechanism underlying the clinical phenotype of CDD. Our finding has revealed a new CDKL5 substrate while also providing a panel of potential CDKL5 substrates for future studies aimed at increasing the definition of the signaling networks in which the protein kinase participates. In addition, our results have shown the first evidence of a new crucial role of CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.