Authors :
Presenting Author: Paul Davies, PhD – Tufts University School of Medicine
Muhammad Arshad, PhD – Tufts University School of Medicine; Stephen Moss, PhD – Tufts University School of Medicine; Jay Mukherjee, PhD – Ovid Therapeutics; Toshiya Nishi, DVM – Ovid Therapeutics; Patrick Sarmiere, PhD – Ovid Therapeutics; Joshua Smalley, PhD – Tufts University School of Medicine
Rationale:
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 epilepsies are a major problem in CDD patients. KCC2 is the principal Cl-extrusion mechanism employed by developing and mature neurons in the CNS. The developmental appearance of hyperpolarizing GABAAR currents is determined by the phosphorylation status of KCC2, a process that facilitates its membrane trafficking and activity. KCC2 loss of function is strongly correlated with cognitive impairment, and the development of pharmaco-resistant seizures that are insensitive to benzodiazepines. To address this issue, we have developed a novel small molecule activator (OV350) that potentiates KCC2 activity.Methods:
KCC2 phosphorylation: Brain lysates prepared from wild-type (WT), or Cdkl5-KO mice (8-12 weeks old) were immunoblotted with; β-actin, CDKL5, KCC2, and phospho-specific antibodies against T906, S940, T1007, and S1022. Kainate (KA)-induced seizures: WT and Cdkl5-KO mice (8 to 10 weeks old) were implanted with electroencephalography/electromyography (EEG/EMG) head mounts and allowed to recover for one week before testing. After a two hour baseline recording, mice were injected with vehicle or OV350 (50 mg/kg i.p.) and monitored for one hour followed by 20 mg/kg i.p. KA injection to induce seizures. Diazepam (5 mg/kg) was administered 30 minutes after entering status epilepticus (SE) and recordings continued for another hour.Results:
Total protein levels of KCC2 did not significantly change between WT and Cdkl5-KO mice, but the phosphorylation of residues S940 and S1022 decreased while T1007 increased in Cdkl5-KO mice compared to WT. In the EEG study, baseline recordings from Cdkl5-KO mice had a significant increase in EEG power compared to WT. Upon KA injections, EEG power of initial seizures, interictal activity, and SE were all significantly increased in Cdkl5-KO mice compared to WT mice. Diazepam failed to fully terminate SE in both genotypes. In the OV350 treated group, baseline EEG power was significantly reduced compared to CDKL5-KO vehicle treated mice. Cdkl5-KO mice treated with OV350 had significantly reduced EEG power of initial seizures, interictal activity, and SE compared to vehicle treated mice. Furthermore, diazepam was able to fully terminate SE.
Conclusions:
The alteration in the phosphorylation of KCC2 in Cdkl5-KO mice (decreased S940, S1022 and increased T1007 phosphorylation) is consistent with described effects resulting from such changes: reduced KCC2 activity, seizures, and cognitive impairment. Treatment with OV350 significantly reduced seizure properties as measured in EEG recordings. OV350 treatment reversed resistance to diazepam and restored its ability to terminate seizure activity in Cdkl5-KO mice. OV350 shows great promise as a therapeutic agent in pharmaco-resistant SE and CDD epileptic encephalopathy.
Funding:
Ovid Therapeutics and NIH NINDS (NS108378, NS111064, NS111338).