Molecular Consequences of Rare Cask Mutations Found in Autism and Neurological Disorders

Background: Differences in synaptic development, pruning, plasticity, and signaling have been implicated in autism spectrum disorder (ASD) both from genetic and functional studies. One of the critical proteins active at the pre- and post-synapse is calcium/calmodulin-dependent serine protein kinase (CASK). The gene is located on the X chromosome, and when mutated causes a range of neurodevelopmental disorders including ASD. In the general population, depletion of loss-of-function and missense mutations affecting CASK have been reported. Although the role of CASK during neuronal development have been studied in model organisms, and it has been shown to interact with many genes involved in ASD etiology in cellular models, studies investigating the direct molecular consequences of CASK mutations in human neurons and how it is involved in ASD etiology are lacking.

Objectives: Our project aims to elucidate the downstream effects of different CASK mutations using patient-derived induced pluripotent stem cell (iPSC) derived neuronal populations.

Methods: We identified two CASK mutation carriers through research and clinical genetic screenings; a male with ASD without cognitive impairments carrying a maternally inherited splice-site mutation and a female with severe intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH) carrying a de novo tandem duplication of two exons. We derived iPSCs from fibroblast of the mutation carriers and two sex-matched controls and analyzed the molecular, morphological and functional consequences of the mutations in iPSC-derived neuronal epithelial stem cells (NES) and differentiated neurons. We performed RNA-sequencing and single cell RNAseq of neuronal populations after 4 week of differentiation to identify dysregulated genes, molecular pathways, and affected neuronal subtypes. Additionally, we analyzed the synaptic and neurite morphology of the neurons and measured individual neuron and neuronal network activity using calcium signaling.

Results: We confirmed in neuronal cells that both mutation carriers have ~50% reduction of CASK protein. This reduction of CASK leads to dysregulation of 560 genes with a false discovery rate <1.0e-5. Among these, downregulated genes converge specifically in the synaptic vesicle cycle pathway and upregulated genes in signaling pathways, such as the Wnt pathway. Our single-cell RNA-seq data suggests that the NES cells in our undirected differentiation protocol develop in to five distinct neuronal cell populations of which the excitatory neuronal population is affected explicitly in neurons derived from the ASD patient. Our preliminary calcium imaging results also suggest that reduced and/or delayed spontaneous network activity occurs in the cells with reduced levels of CASK.

Conclusions: Our results indicate that rare variants in CASK impair the synaptic vesicle pathway with consequences to neuronal activity. Moreover, our results highlight potential drug targets such as the Wnt signaling pathway which could potentially act as a route for drug development for CASK deficiency. The use of iPSC-derived neurons from mutation carriers with variable phenotypes opens a window to understanding direct consequences of CASK mutations at the molecular and single cell level.