SOLVING THE PUZZLE OF PROTOCADHERIN-19 MOSAICISM TO UNDERSTAND THE PATHOPHYSIOLOGY OF DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 9 (DEE9)

Developmental and Epileptic Encephalopathy 9 (DEE9) is a severe neurological disorder characterized by clustered epilepsy, intellectual disability (ID) and autism spectrum disorder (ASD) (Dibbens et al., 2008). DEE9 is caused by mutations affecting the X – linked gene PCDH19, which encodes for a calcium – dependent cell – cell adhesion molecule, called protocadherin – 19 (PCDH19) (Dibbens et al., 2008). PCDH19 is mainly expressed in the Central Nervous System (CNS), where it is involved in cell – adhesion, neuronal migration, and circuit formation (Cooper et al 2015). Even though DEE9 is a X – linked disorder, the 90% of the patients are females (Shibata et al., 2021). This peculiarity was attributed to a cellular interference mechanism: due to random chromosome X inactivation, female patients have a mosaic expression of PCDH19 in the brain. This mosaicism is supposed to be responsible for a scrambled neuronal communication, promoting the onset of DEE9 features (Dibbens et al., 2018). The cellular interference hypothesis was supported by the identification of few DEE9 male patients with PCDH19 somatic mutations (Niazi et al., 2019). However, pathophysiological mechanisms behind DEE9 are still unclear and the generation of animal models could help in elucidating them. In our laboratory, we generated a new conditional knock – out (cKO) mouse model for PCDH19, through the Cre – Lox P technology (Pcdh19 floxed mouse). Two different approaches were used to deliver Cre recombinase: 1) crossbreeding of Pcdh19 floxed mice with mice expressing Cre under the rat Synapsin – 1 promoter, to target specifically neurons; 2) intracerebroventricular (ICV) injection in Pcdh19 floxed mice of an adeno – associated virus (AAV) expressing Cre fused to GFP. This last approach allowed to discriminate PCDH19 positive from PCDH19 negative neurons. Once evaluated in vitro specific Cre – mediated excision and absence of protein production by activation of the Nonsense Mediated Decay (NMD) system, we molecularly, functionally, and behaviorally characterized the new Pcdh19 cKO mouse model. In cortical and hippocampal tissues, Pcdh19 cKO female mice were characterized by a reduction in both PCDH19 mRNA and protein of 40% compared to control female mice. Interestingly, also Pcdh19 cKO male mice were mosaic for PCDH19 expression, most likely due to the low Cre expression under the relatively weak Synapsin – 1 promoter. Indeed, they displayed a mRNA and protein reduction of 60% compared to their sex – related controls. So, both Pcdh19 cKO female and male mice recapitulated PCDH19 brain mosaicism, considered DEE9 triggering feature. This allowed us to perform some of the analyses on both sexes, to identify a possible gender effect associated to DEE9. Pcdh19 cKO female mice were characterized by synaptic defects in the hippocampal CA1 region. Indeed, they showed a reduced number of excitatory synapses with a reduced number of neurotransmitter vesicles and reduced post-synaptic density (PSD) thickness compared to control female mice. In association with synaptic structural defects, Pcdh19 cKO female mice presented also impaired synaptic functionality. Indeed, Pcdh19 cKO female mice were characterized by a reduced Long-Term Potentiation (LTP) and a reduced Paired Pulse Ratio (PPR) compared to their sex – matched control mice. These synaptic defects prompted us to investigate the behavioral features of Pcdh19 cKO mice. Since DEE9 is characterized by ID and ASD, we investigated these two aspects. Pcdh19 cKO female and male mice displayed an increased number and duration of self – grooming events, suggesting an ASD-like phenotype. Moreover, Pcdh19 cKO mice of both sexes showed impairment in learning and memory plasticity, evaluated through the Morris Water Maze (MWM) test. Interestingly, the Fear Conditioning Test reconfirmed hippocampal – related memory defects exclusively in female cKO, suggesting that the female sex could be more susceptible to Pcdh19 loss. Concerning epilepsy, our Pcdh19 cKO mouse model didn’t show spontaneous seizures, as observed in the constitutive Pcdh19 KO mouse models (Pederick et al., 2016, Hoshina et al., 2021). However, Pcdh19 cKO mice displayed some hyperexcitability features at subclinical level. Indeed, PCDH19 negative neurons in the mosaic brain of Pcdh19 floxed mice were characterized by a reduced rheobase and by a higher firing frequency compared to neighboring cells retaining PCDH19 expression. Moreover, Pcdh19 cKO mice were characterized by an aberrant surface expression of the GABAARs 1 subunit, underlying possible GABAergic defects. To conclude, we generated a new Pcdh19 cKO mouse model which was able to recapitulate Pcdh19 brain mosaicism and features of ID and ASD, as in DEE9 pathology. Besides behavioral alterations, also functional and morphological synaptic defects in hippocampus were noticed. Finally, our mouse model provided clues of a GABAergic impairment and a possible gender – effect at the basis of DEE9 pathophysiology.