Evidence is accumulating that some childhood-onset epilepsies arise from presynaptic defects (1). The presynapse releases chemical neurotransmitters in response to action potential stimulation and this event is sustained by synaptic vesicle (SV) endocytosis. Mutations in the gene encoding the protein kinase cyclin-dependent kinase-like 5 (CDKL5) result in CDKL5 deficiency disorder (CDD). We have important unpublished data demonstrating that SV endocytosis is specifically disrupted in CDKL5 knockout neurons and that the protein kinase activity of CDKL5 is essential for this role. Therefore, CDKL5 phosphorylates an unidentified presynaptic substrate to control SV endocytosis at the presynapse.
The project aims to
Aim 1) A phospho-proteomic screen of CDKL5 knockout neurons has been performed and has identified potential presynaptic CDKL5 substrates and their phosphorylation sites. These will be validated by western blotting with either phospho-specific antibodies or immunoprecipitation of candidates followed by probing with anti-Ser/Thr antibodies. Rescue experiments will be performed via shRNA-mediated depletion of the endogenous candidates and co-expression of exogenous phospho-null or –mimetic forms in primary cultures of both wild-type and CDKL5 knockout neurons. The student will also have the opportunity to determine how CDKL5-dependent phosphorylation of these candidates affects their molecular interactions by performing affinity chromatography from neuronal lysates with phospho-null and –mimetic candidates.
Aim 2) The student will choose to investigate the physiological role of CDKL5-dependent phosphorylation on either neurotransmission (using slice electrophysiology) or established behavioural defects via viral delivery of phospho-null and –mimetic forms of the candidate into the hippocampus of either wild-type or CDKL5 knockout rats.
Therefore, the student will be trained in state-of-the-art molecular neuroscience techniques (primary neuronal culture, generation of molecular tools, live cell fluorescence imaging acquisition and analysis, determination of interaction partners via GST-pull downs and immunoprecipitation, virus design and delivery, slice electrophysiology / behavioural analysis) to determine the role of a key epilepsy gene, CDKL5, in synaptic, circuit and behavioural function.
