Abstract. Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a neurodevelopmental epileptic
encephalopathy that is characterized by early-onset epilepsy, sleep disturbances, and developmental
challenges. Pharmaco-resistant epilepsy with generalized, multifocal, and myoclonic seizures are a major issue
for CDD patients. Currently, no targeted treatment or gene therapy exists for CDKL5 disorder. CDKL5 is a
serine/threonine protein kinase and although it is known to be essential for normal brain development and
function it is unknown which proteins are targeted by CDKL5. KCC2 is the principal Cl--extrusion mechanism
employed by developing and mature neurons in the CNS. Its activity is a prerequisite for the efficacy of fast
synaptic inhibition mediated by ¿-aminobutyric acid type A receptors (GABAAR), which are Cl- permeable ligand-
gated ion channels. The postnatal development of canonical hyperpolarizing GABAAR currents reflects the
progressive decrease of intraneuronal Cl- levels that is caused by the upregulation of KCC2 expression and
subsequent activity. The developmental appearance of hyperpolarizing GABAAR currents is determined by the
phosphorylation status of KCC2, a process that facilitates its membrane trafficking and activity. Deficits in KCC2
expression levels and activity have been detailed in patient and animal models of epilepsy. Furthermore, we
have demonstrated that KCC2 loss of function is strongly correlated with cognitive impairment, and the
development of pharmaco-resistant seizures that are insensitive to GABAAR positive allosteric modulators such
as benzodiazepines. To address this issue, we have developed novel small molecule activators that potentiate
KCC2 activity which, in preliminary studies, have been shown to effectively terminate pharmaco-resistant
seizures. CDKL5 null mice have seizure-like EEG events at postnatal day 12 and had a significant decrease in
the amount of phosphorylated KCC2. We will further examine this relationship between KCC2-S1022
phosphorylation and CDKL5. Our working hypothesis is:Loss of CDKL5 results in a reduced phosphorylation
of S1022 KCC2, decreased KCC2 activity, and an increased susceptibility to pharmacoresistant seizures.
Small molecule activators of KCC2 will increase KCC2 activity and prevent pharmacoresistance
seizures. To test this hypothesis, we propose the following aims: Aim1. To determine the effects of ablating
CDKL5 expression on KCC2 phosphorylation. Aim 2. To determine the effects of ablating CDKL5
expression on KCC2 activity. Specific Aim 3. Determine the consequences of ablating KCC2 S1022
phosphorylation on KCC2 function and expression. The result of this study will provide new insights into the
targets of CDKL5 and the mechanisms that result in the debilitating pharmaco-resistant epilepsy occurring in
CDD. Such insights may promote the development of new therapeutics to alleviate the burdens of pharmaco-
resistant epilepsies that affect many epileptic patients.
