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CASK mutation Disrupts Neural Connectivity in Human Neurons

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE199910 | GEO | 2022/04/03

REPOSITORIES: GEO

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Project description:Background: Altered neuronal development is discussed as underlying pathogenic mechanism of Autism Spectrum Disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during in-vitro neuronal differentiation. We hypothesized a causal relation between this tryptophan related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on differentiation of a neuronal cell line and specifically focused on neuronal morphology, metabolites of the tryptophan pathway and the neurodevelopmental transcriptome. Methods: The gene dosage dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical inhibition, and (iii) complete CRISPR/Cas9 induced knockout (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain. Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with neurite maturation in SH-SY5Y cells. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT associated tryptophan pathway was not affected by KO. At the transcriptome level neuronal processes like “synapse organization” were affected. Differentially regulated genes were enriched for ASD-candidates and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala. Conclusions: In this study QPRT was causally related to in vitro neuronal differentiation and affected the regulation of genes and gene-networks that were previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.

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CASK mutation Disrupts Neural Connectivity in Human Neurons

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