Analysing schizophrenia risk variants in neurexin 1 using functional and mature neuronal cultures from patient-derived induced pluripotent stem cells

Introduction:

There is a growing interest in psychiatry to connect causal genetic variants to neurobiological dysfunction. Genomic studies indicate that copy number variants (CNVs) are related to the development of schizophrenia. Schizophrenia is a complex psychiatric disorder that affects nearly 1% of the world's population. We previously described deletions in neurexin 1 (NRXN1) to be associated with schizophrenia. Neurexins are transcribed from of three different genes in mammalians (NRXN1, NRXN2, and NRXN3) and are widely expressed in excitatory as well as inhibitory neurons, in particular in presynaptic terminals required for normal synapse function. Neurexins are neuronal adhesion molecules interacting with neuroligins. Disruption of the NRXN1 leads affects properties of synapses and leads to the disruption of neuronal networks. Especially, deletions in α-neurexin 1 are involved in altered neural connectivity.

Methods:

For further analysis of NRXN1-related disease mechanisms, we used an in vitro cell culture model based on human induced pluripotent stem cells (iPS cells). Human iPS cells have been obtained by reprogramming of B-lymphoblastoid cell lines (B-LCLs) that have been obtained from schizophrenia patients carrying CNVs in NRXN1. In one case the CNV is located in promotor region and exon 1 – 6 and in the other case carries a CNV in exon 4 – 6. Patient derived induced pluripotent stem cells and healthy control cells were differentiated into mature and functional cortical neurons. The analysis focused on gene expression regulating signaling pathways, which are part of the NRXN1 network. We applied IF analysis, WB analysis, flow cytometry, and metabolic phenotyping.

Results:

Gene expression analysis confirmed the successful generation of mature glial and neuronal cells (TUBB3, STX, GFAP, and CD68). The presence of different neuronal subtypes such as GABAergic and glutamatergic neurons was demonstrated by the induction of crucial markers including SLC17A7, GAD1, GABBR1, GRIA2, and GRIN1. IF analysis verified the presence of NRXN1 and its interaction partners. We found differently regulated NRXN1 interaction partners in iPS cells carrying the CNV in NRXN1 and we also found differences in the metabolism of iPS cell-derived neurons.

Conclusion:

In summary, the characterization of iPS cells carrying a patient-specific genetic information and their differentiation into schizophrenia-specific neurons has a high potential to elucidate the impact of specific genetic variations in NRXN1. Such in vitro models represent promising models for applications using screening platforms enabling the identification of potential therapeutic targets in schizophrenia.