CDKL5 is a protein kinase that plays a key role for neuronal functions as testified by the onset of complex neuronal dysfunctions in patients with genetic lesions in CDKL5. Here we identify a novel interactor of CDKL5, IQGAP1, a fundamental regulator of cell migration and polarity. In accordance with a functional role of this interaction, depletion of CDKL5 impairs cell migration and impedes the localization of IQGAP1 at the leading edge. Moreover, we demonstrate that CDKL5 is required for IQGAP1 to form a functional complex with its effectors, Rac1 and the microtubule plus end tracking protein CLIP170. These defects eventually impact on the microtubule association of CLIP170, thus deranging their dynamics. CLIP170 is a cellular target of the neurosteroid pregnenolone; by blocking CLIP170 in its active conformation, pregnenolone is capable of restoring the microtubule association of CLIP170 in CDKL5 deficient cells and rescuing morphological defects in neurons devoid of CDKL5. These findings provide novel insights into CDKL5 functions and pave the way for target-specific therapeutic strategies for individuals affected with CDKL5-disorder.

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Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a complex neurological disorder, characterized by infantile seizures, impairment of cognitive and motor skills and autistic features. Loss of Cdkl5 in mice affects dendritic spine maturation and dynamics but the underlying molecular mechanisms are still far from fully understood. Here we show that Cdkl5 deficiency in primary hippocampal neurons leads to deranged expression of the alpha-amino-3-hydroxy-5-methyl-4-iso-xazole propionic acid receptors (AMPA-R). In particular, a dramatic reduction of expression of the GluA2 subunit occurs concomitantly with its hyper-phosphorylation on Serine 880 and increased ubiquitination. Consequently, Cdkl5 silencing skews the composition of membrane-inserted AMPA-Rs towards the GluA2-lacking calcium-permeable form. Such derangement is likely to contribute, at least in part, to the altered synaptic functions and cognitive impairment linked to loss of Cdkl5. Importantly, we find that tianeptine, a cognitive enhancer and antidepressant drug, known to recruit and stabilise AMPA-Rs at the synaptic sites, can normalise the expression of membrane inserted AMPA-Rs as well as the number of PSD-95 clusters, suggesting its therapeutic potential for patients with mutations in CDKL5.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a neurodevelopmental disease characterized by severe infantile seizures and intellectual disability. The absence of CDKL5 in mice causes defective spine maturation that can at least partially explain the cognitive impairment in CDKL5 patients and CDD mouse models. The molecular basis for such defect may depend on the capacity of CDKL5 to regulate microtubule (MT) dynamics through its association with the MT-plus end tracking protein CLIP170. Indeed, we here demonstrate that the absence of CDKL5 causes CLIP170 to be mainly in a closed inactive conformation that impedes its binding to MTs. Previously, the synthetic pregnenolone analogue, pregnenolone-methyl-ether (PME), was found to have a positive effect on CDKL5-related cellular and neuronal defects in vitro. Here we show that PME induces the open active conformation of CLIP170 and promotes the entry of MTs into dendritic spines in vitro. Furthermore, the administration of PME to symptomatic Cdkl5-knock-out mice improved hippocampal-dependent behavior and restored spine maturation and the localization of MT-related proteins in the synaptic compartment. The positive effect on cognitive deficits persisted for one week after treatment withdrawal. Altogether, our results suggest that CDKL5 regulates spine maturation and cognitive processes through its control of CLIP170 and MT dynamics, which may represent a novel target for the development of disease modifying therapies.