Fellowship for S. Schwarz: Mechanism of Altered Synaptic Signaling by Disease Related GRIN1 Variants

PROJECT SUMMARY / ABSTRACT Variations in the GRIN1 gene result in neurological disorders with an incidence rate of 5.45 per 100,000 births. Among these rare diseases, symptoms include epilepsy and seizures, intellectual disabilities and developmental delays, and behavioral and movement disorders. The GRIN1 gene encodes the obligatory GluN1 subunit of N- methyl-D-aspartate receptors (NMDARs), which are postsynaptic ligand-gated ion channels that are essential in the standard development and operation of the central nervous system. Pathological variations in this gene often occur in and around the critical activation gate of the receptor, which controls the opening and closing of the ion channel pore. Of the many pathologic GRIN1 variations identified so far, only a handful have been functionally characterized, and GRIN1-related disorders remain poorly understood. This project will delineate the activity, gating mechanism, and potential pharmacological interventions of two pathological GluN1 variations at residue 647 (Y647C and Y647S). Patients with either of these variants exhibit severe cognitive developmental delay, acquired microcephaly, extensive bilateral polymicrogyria, and seizure activity. The experiments proposed will test the HYPOTHESIS that the GluN1 Y647C and Y647S variants impair receptor signaling by producing gating deficits, which may be rescued with positive allosteric modulators. APPROACHES will include protein chemistry and mutagenesis, molecular biology, and patch-clamp electrophysiology in cultured cells and mouse brain slices. Aim 1 will characterize biophysical properties and disrupted inter-subunit contacts of recombinant NMDARs containing the GluN1 Y647C and Y647S variants in human embryonic kidney (HEK293) cells. Aim 2 will examine pharmacological rescue of these variants with positive allosteric modulators of NMDARs, GNE-9278 and GNE- 0723. Aim 3 will investigate how the GluN1 Y647S variant affects synaptic transmission in both excitatory and inhibitory neurons and the effect of pharmacological interventions using brain slices from mice with the same genotype as human patients (GRIN1+/Y647S). This project will RESULT in a comprehensive characterization of two pathological GRIN1 variants and will provide a greater understanding of the disease mechanism in GRIN1- related disorders. Additionally, this project will study how these variants respond to NMDAR modulators with the goal of rescuing their hypofunction. The approaches leveraged here may serve to study other GRIN1 variants to expand our understanding of NMDARs in health and disease and may help with informing and developing individual patient treatments depending on the variant they carry.