DPYSL2/CRMP2isoform B knockout in human iPSC-derived glutamatergic neurons confirms its role in mTOR signaling and neurodevelopmental disorders

Abstract

DPYSL2/CRMP2 is a microtubule-stabilizing protein crucial for neurogenesis and associated with numerous psychiatric and neurodegenerative disorders. DPYSL2 has multiple RNA and protein isoforms, but few studies have differentiated between them or explored their individual functions. We previously demonstrated in HEK293 cells that a schizophrenia-associated variant in the DPYSL2 B isoform (DPYSL2-B) reduced the length of cellular projections, created a transcriptomic disturbance that captured schizophrenia etiology, and was acted upon by the mTOR pathway. In the present study, we follow up on these results by creating, to our knowledge, the first models of endogenous DPYSL2-B knockout in human induced pluripotent stem cells and excitatory glutamatergic neurons. We use CRISPR/Cas9 to specifically knock out DPYSL2-B and observe corresponding reduction of its RNA and protein. The average length of dendrites in knockout neurons was reduced up to 58% compared to controls. Transcriptome analysis reveals disruptions in pathways highly relevant to psychiatric disease including mTOR signaling, cytoskeletal dynamics, immune function, calcium signaling, and cholesterol biosynthesis. We also observed a significant enrichment of our differentially expressed genes in schizophrenia GWAS-associated loci. Our findings clarify the functions of the human DPYSL2-B isoform and confirm its involvement in molecular pathologies shared between many psychiatric diseases.

Insights for disease modeling from single-cell transcriptomics of iPSC-derived Ngn2-induced neurons and astrocytes across differentiation time and co-culture

“…Immunofluorescence images of the Ngn2 induced neurons can be seen in Additional file 26: Sup. Figure 17 and our previous publications [59][60][61].…”

Distinctivein vitrophenotypes in iPSC-derived neurons from patients with gain- and loss-of-functionSCN2Adevelopmental and epileptic encephalopathy

“…NLRP2 is altered in opposite directions in the two models (increased expression in R1882Q and decreased in R853Q), whereas CHL1 is upregulated in both. Dysregulation of NLRP2 , involved in the regulation of immune responses, has been observed in familial early-onset Alzheimer’s (48), late-onset Alzheimer’s (49,50), and in bipolar disorder (51,52). Deletions of CHL1 have been described in patients with neurodevelopmental delay and seizures, but less is known about the potential effect of CHL1 overexpression.…”

A Functional Schizophrenia-associated genetic variant near theTSNARE1andADGRB1genes

“…Cellular modeling is quickly becoming a powerful tool for the study of genetic risk variants, particularly those within non-coding regions of the genome, as technological advances are being made in genome editing, iPSC generation from somatic cells, and directed differentiation methods 22 . Now, we are able to generate, study, and compare human neuronal cells with only a single nucleotide difference across the entire genome, as we and others have previously reported 21,29,[66][67][68][69][70] . This approach provides the opportunity to elucidate the individual contributions of risk variants in a human-and disease-relevant system.…”