Abstracts

Rationale: PCDH19-related epilepsy is an early infantile epileptic encephalopathy characterized by brief and repeating seizure clusters. PCDH19-related epilepsy shows a unique pattern of X-linked inheritance in which heterozygous females present with seizures, while hemizygous carrier males are asymptomatic. It is speculated that the disease mechanism follows cellular interference: the co-existence of two cell populations, cells with wild-type PCDH19 and cells with mutant PCDH19 expression, arising from random X-inactivation in heterozygous females, disrupt proper homophilic binding between cells, leading to epilepsy. Recently, PCDH19 has been shown to be a potential modulator of GABAergic transmission by interacting with the GABAA receptor (GABAAR) alpha subunit. Given the importance of GABAAR -mediated transmission in epileptogenesis and cognitive impairment, we used two different acute seizure tests that target GABAAR -mediated signaling to characterize seizure susceptibility in the PCDH19 mouse model.  Methods: We induced generalized seizures using a noncompetitive GABAAR antagonist, flurothyl. 8-12-week-old PCDH19-/- (n=9), PCDH19-/+ (n=10), and female WT (n=10) mice were administered flurothyl (2,2,2-trifluroethyl ether). Latencies from flurothyl induction to the first myoclonic jerk (MJ), first generalized tonic-clonic seizure (GTCS), and hindlimb extension (HLE) were recorded. At the onset of GTCS, the chamber top was removed and the mouse was immediately placed in a holding cage for recovery.We next induced focal seizures using a 6-Hz electroshock behavioral paradigm, which has previously been suggested to target GABAAR. 6-Hz psychomotor seizures were induced in 8-12-week-old PCDH19-/- (n=13), PCDH19-/+ (n=20), and female WT (n=18) mice via auricular stimulation (0.2 ms pulse width at 6 Hz, 3 s duration). All genotypes were shocked at current intensities of 16, 24, and 30 mA and scored according to a modified Racine scale. Animals that did not resume normal exploratory behavior within 10 seconds following stimulation were not tested at higher current intensities.  Results: Upon flurothyl administeration, PCDH19-/- and PCDH19-/+ mice showed significantly less time to myoclonic jerk onset compared to their WT counterparts (p= 0.0087, p= 0.0451). The average latencies to generalized tonic-clonic seizure (GTCS) and hindlimb extension (HLE) were comparable among all genotypes.In the 6-Hz electroshock behavioral paradigm, PCDH19-/- and PCDH19-/+ mice showed significantly more robust seizure phenotypes compared to their WT littermates at low intensity stimulation (p= 0.0003, p= 0.0265). At a current level of 24 mA, PCDH19-/- and PCDH19-/+ mice showed significantly increased seizure severity compared to their WT littermates (p= 0.0059, p= 0.0004). By 30 mA, most of the PCDH19-/- and PCDH19-/+ mice had seized and were subsequently removed from the experiment, while the WT mice showed a range of seizure severity.  Conclusions: Since PCDH19 and GABAAR have been shown to interact, we performed two seizure tests in the PCDH19-epilepsy mouse model that have been previously implicated in affecting GABAAR signaling. Our results show that PCDH19-/- and PCDH19-/+ female mice exhibit increased seizure susceptibility to both flurothyl-induced and electroshock-induced seizures. To our surprise, the increased seizure susceptibility and severity of PCDH19-/- mice did not reflect the mechanism of cellular interference, which requires PCDH19 mosaicism for the disease phenotype. Our results suggest that pathogenic mechanisms in addition to cellular interference should be considered in future studies. Funding: No funding