CASK mutation Disrupts Neural Connectivity in Human Neurons

Loss-of-function mutations in CASK cause severe developmental phenotypes including microcephaly with pontine and cerebellar hypoplasia (MICPHC), X-linked intellectual disability and autism. Modeling of CASK-related disorders has been challenging due to limited human cellular models to study the multifaceted roles of this protein during neuronal and synapse development. Here, we generated two independent CASK knockout (KO) isogenic cell lines from human embryonic stem cells (hESCs) using CRISPR/Cas9 and performed a detailed characterization of gene expression, morphology and neuronal function during neuronal differentiation and synapse development. While immature neurons showed an upregulation of gene networks related neuronal cell adhesion, projection and outgrowth, concomitant with increased neuronal complexity in CASK KO neurons, mature neurons displayed severe defects in neuronal spiking, spontaneous synaptic transmission and synchronized burst firing patterns without significant changes in neuronal morphology and synapse numbers. In developing human cortical neurons, CASK functions to promote both structural integrity and establishment of cortical excitatory neuronal networks. These results lay the foundation for future studies identifying suppressors of such phenotypes relevant to human patients. Day 7 human cortical excitatory neurons were collected for bulk RNAseq from 4 independent culture replicates of WT, CASK KO#1 and CASK KO#2 hESC lines. Day 28 human cortical excitatory neurons cultured with mouse glia were also collected for bulk RNAseq from 4 independent culture replicates of WT, CASK KO#1 and CASK KO#2. Differential gene expression analysis was carried out.