Modeling schizophrenia with patient-specific mutations in GRIN2A and SP4

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PROJECT SUMMARY: Schizophrenia (SCZ) is a common and debilitating mental illness characterized by positive symptoms, negative symptoms, and impaired cognition, with a lifetime prevalence approaching 1% in the United States. Unfortunately, progress in understanding the etiology and pathophysiology is hindered by the lack of appropriate models that adequately capture both the complex and heterogeneous nature of the genetic risk and the diversity of the phenotypic manifestations. The advent of induced pluripotent stem cell (hiPSCs) technology provides an important new platform to study the cellular and circuit behavior of human cells that contain an individual’s full complement of primary sequence risk alleles. Thus, the door is now opened to developing improved in vitro models that can enhance our understanding of etiology and pathophysiology in an integrated context, relating patient genomes to their cellular phenotypes. Here, we propose to generate novel hIPSC lines harboring patient-specific mutations in two highly penetrant SCZ risk genes, GRIN2A and SP4. GRIN2A encodes a subunit of the N-methyl-D-aspartate receptor (NMDAR) which are a subclass of ionotropic glutamate receptors that play a pivotal role in the development, plasticity, and pathophysiology in the central nervous system. NMDAR hypofunction is a leading causal hypothesis for SCZ because subanesthetic doses of the NMDAR antagonists produces acute psychosis in healthy subjects. In addition, autoimmune encephalitis due auto-immunoreactivity to NMDARs leads to severe psychosis. SP4 belongs to the SP1 family of transcription factors that recognizes DNA sequences termed GC-boxes that are often found upstream of transcription start sites (TSS). SP4 has been shown to functionally regulate the expression of NMDARs through direct interactions with GC-boxes upstream of the TSS of GRIN1, GRIN2A, and GRIN2B. In addition, Sp4 hypomorphic mice have reduced hippocampal long-term potentiation, reduced NMDAR expression, and behavioral abnormalities related to SCZ. Importantly, both GRIN2A and SP4 are high priority SCZ risk genes, being associated with SCZ through genome-wide association studies and rare variant analysis. We propose to use CRIPSR editing to generate patient-specific mutations in GRIN2A and SP4 with consideration of the genetic background of the hIPSC lines. Both genes will be individually edited in the same six control hiPSCs lines with elevated SCZ polygenic risk scores. We will perform quality control experiments to confirm mutations are correct and no off-target mutations are present. Validation of functional mutations will be confirmed with qPCR, Western blot and electrophysiology. Once validated, we will quantify the effect of these mutations on spontaneous network activity using multi-electrode arrays and their effect on the transcriptome with RNA sequencing. The overall goal of this proposal is to develop novel cellular models of SCZ that can inform us about pathophysiology and help identify convergent molecular mechanism in SCZ with the goal of identifying therapeutic targets for this debilitating disorder.