Authors :
Presenting Author: Miranda Walker, BS – University of Michigan
Wei Niu, Ph.D. – Research Assistant Professor, Neurology, University of Michigan; Jack Parent, M.D. – Professor, Neurology and Michigan Neuroscience Institute, University of Michigan; Michael Uhler, Ph.D. – Professor, Biological Chemistry and Michigan Neuroscience Institute, University of Michigan
Rationale: Protocadherin-19 (PCDH19) Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy (DEE) characterized by early childhood intractable seizure clusters, impaired motor and cognitive development, and neuropsychiatric comorbidities. PCE is caused by loss-of-function (lof) variants in the X-linked gene
PCDH19, which encodes for a transmembrane cell adhesion molecule important for brain development. PCE affects heterozygous females and mosaic males but spares hemizygous males. In heterozygous females, random X-inactivation leads to mosaic populations of neurons expressing only wild-type (WT) or only mutant
PCDH19. Impaired cell-adhesion between these two populations is hypothesized to cause abnormal cell segregation and alter cortical development; however, the mechanisms of how
PCDH19 mosaicism causes seizures are not fully understood. Additionally, although interneuron dysfunction is implicated in many DEEs, the role of PCDH19 in cortical interneuron development remains relatively unexplored. Here, we use 2D neuronal models to study the mechanisms through which
PCDH19 lof gene variants lead to cortical network hyperexcitability and seizures.
Methods: We generated induced excitatory neurons (iNeurons) and induced GABAergic neurons (iGN) from PCDH19 WT and CRIPSR knock-out (KO) H9 human embryonic stem cells (hESCs) through doxycycline-inducible expression of the human transcription factors NGN2 or ASCl1 and Dlx2, respectively. Neurons were cultured for one to seven weeks, either alone or in co-cultures of both iNeurons and iGNs. We assessed differences in migration, synapse formation, neurite morphology, gene expression, and neuronal excitability using immunostaining, RT-qPCR, and calcium Imaging methods.
Results: Compared to WT iGNs, we found PCDH19 KO iGNs exhibit altered migration, decreased expression of the pre-synaptic marker vGAT and changes in neurite morphology. In agreement with findings in mouse models, our KO iNeurons show decreased excitability and network synchrony. Additionally, KO iGNs were less effective than WT iGNs at inhibiting WT iNeurons when grown in co-culture.
Conclusions: Our 2D human neuronal models reveal differences in migration, network excitability and neuronal morphology, suggesting that PCDH19 is important for interneuron development and functional synapse formation in human excitatory and inhibitory cortical neurons. Further study of these models should provide novel insights into PCDH19 function and mechanisms underlying PCE.
Funding: NIH-NIGMS Training Program in Translational Research pre-doctoral fellowship 5T32GM113900-05 (MW), U54 NS117170 (JMP), and SFARI (WN and JMP).