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Title: Novel CDKL5 substrates and functions in neurodevelopment
Author: Baltussen, Lucas L.
ISNI:       0000 0004 7429 1103
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Cyclin-Dependent Kinase-Like 5 (CDKL5) is a serine/threonine protein kinase important for neuronal development. Mutations in the CDKL5 gene are responsible for CDKL5 deficiency disorder, a rare neurodevelopmental disorder displaying a heterogeneous range of clinical phenotypes. CDKL5 is enriched in the brain during early postnatal development, and is known to be involved in the development of dendritic spines and synapses. However, direct downstream effectors of CDKL5 and its molecular mechanisms of action remain unknown. We have generated analogue-specific CDKL5 by mutating the gatekeeper residue in the ATP-binding pocket, and introduced a second-site mutation to rescue kinase activity. Based on the utilization of bulky ATP analogues by analogue-specific CDKL5, we used an unbiased chemical genetic screen to identify CDKL5 substrates and phosphorylation sites in mouse brain. In vitro validation of ARHGEF2, EB2 and two sites in MAP1S revealed RPxS as a common CDKL5 phosphorylation motif. We show that EB2 and MAP1S phosphorylation is strongly reduced in brains of Cdkl5 KO mice. In neurons derived from CDKL5 patient iPSCs, we observe similar reductions indicating that regulation of these phosphorylation sites is conserved in humans. By using CDKL5 substrate phosphorylation as a read-out, we have been able to show that CDKL5 activity is regulated during brain development and affected by neuronal activity. These novel CDKL5 substrates share microtubule binding properties, but only phosphorylation of MAP1S by CDKL5 is directly regulating its microtubule binding affinity. Attempts to identify a molecular function of EB2 phosphorylation did not lead to satisfying results. Both MAP1S and EB2 are known to be involved in microtubule dynamic instability, the process of constant growth and collapse of microtubule plus-ends. We show that primary cortical cultures of Cdkl5 KO mice have altered microtubule plus-end dynamics visualised by sparser, but longer EB3 comets. Knockdown of MAP1S, but not EB2, partially rescued this phenotype in Cdkl5 KO neurons, indicating a downstream function of MAP1S in the regulation of microtubule dynamics.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available