Impedance-based phenotypic profiling of metabotropic glutamate receptor ligand responses in SCA1 human iPSC-derived neuronal cultures

Abstract

Group 1 metabotropic glutamate receptors (mGluRs) are a family of G protein-coupled receptors (GPCRs) including mGluR1 and mGluR5. These receptors are expressed throughout the central nervous system and play an important role in synaptic plasticity. Dysfunction of mGluR1 in cerebellar Purkinje cells (PC) is observed in spinocerebellar ataxia type 1 (SCA1), an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in the ATXN1 gene. MGluR1 dysfunction disrupts PC signaling and ultimately contributes to PC death and overall progressive cerebellar dysfunction observed in SCA1. To investigate mGluR1/5 pharmacology in the context of SCA1, mGluR1/5 function was assessed in SCA1 and control human induced pluripotent stem cell (hiPS)cell-derived neuronal cultures using impedance measurements in the xCELLigence real-time cell analyzer (RTCA) system. Culture conditions and protocols for evaluating glutamate receptor activity were first optimized. Subsequently, GRM1 and GRM5 expression levels were assessed and glutamatergic signaling function was characterized in SCA1 hiPS cell-derived neuronal cultures using both the non-selective endogenous ligand L-glutamate and the selective orthosteric agonist for mGluR1/5, (RS)-3,5-Dihydroxyphenylglycine. To specifically characterize mGluR1 function, mGluR1-specific positive- and negative allosteric modulators (Ro0711401 and JNJ16259685 respectively) were tested. Results showed reduced mGluR1 and decreased mGluR5 RNA expression levels and diminished mGluR1/5 responses in the SCA1 hiPS cell-derived neuronal cultures. These results underline the potential utility of impedance measurements for characterizing GPCR function and pharmacological testing in a high-throughput manner in patient-derived neuronal cultures.