LAUSR

Background Schizophrenia is a neurodevelopmental disease. Causes include environmental and genetic factors. Characteristics of schizophrenia include a deregulation of the glutamatergic and the GABAergic neurotransmission and the degeneration of predominantly inhibitory interneurons. Recently, 108 loci referring to Single Nucleotide Polymorphisms (SNPs) and 8 loci containing Copy Number Variations (CNVs) have been associated with schizophrenia suggesting a strong impact of genetic factors on the embryonic development of human brain. Together with other research teams, we identified CNVs in the Neurexin 1 gene (NRXN1) in patients suffering from schizophrenia, which highly recommends the detailed analysis of NRXN1-related disease mechanisms. Neurexins are presynaptic transmembrane proteins. We established patient-specific induced Pluripotent Stem (iPS) cells from schizophrenia patients and healthy donors to differentiate neurons for the investigation of disease mechanisms related to NRXN1 in schizophrenia patients. Patient-specific iPS cells carry the disease-associated genetic background of a patient, which is a CNVs affecting NRXN1. Methods Human iPS cells were generated from B-Lymphoblastoid Cell Lines (B-LCLs) using episomal vectors. B-LCLs were obtained from patients carrying CNVs in NRXN1 and healthy donors. The pluripotency of stem cells was verified by alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers. A neuronal screening protocol was applied to select iPS cell clones with a high differentiation potential towards neuronal cell lineages. Established lines were differentiated into Neural Stem Cells (NSCs) and further differentiated into functional and mature neurons. Terminal differentiated neurons were characterized by morphology, transcript analysis, western blot analysis, immunofluorescence analysis, electrophysiology, and mitochondrial respiration. Results Transcript analysis and western blot analysis of iPS cells verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The induction of neural stem cells was characterized by mRNA and protein analysis of neural stem cells markers including SOX2 and PAX6. Neural induction towards cortical neurons was monitored by a set of neurodevelopmental marker genes including NESTIN, GFAP, and NGN3. The efficient differentiation of mature neurons was shown by the expression of marker genes including glutamate and GABA receptors. Mitochondrial respiration was elevated in differentiated neurons. Patch clamp analysis verified the differentiation of functional GABAergic and glutamatergic neurons. Discussion Human iPS cells provide a powerful tool for the analysis of schizophrenia-associated and patient-specific DNA variations including CNVs. We successfully established mature and functional neuronal cultures suitable to study disease mechanisms related to NRXN1. Accordingly, this approach is an excellent opportunity for the identification of potential therapeutic targets.