Complex regulation of Gephyrin splicing is a determinant of inhibitory postsynaptic diversity

Abstract

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and gamma-aminobutyric acid A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.

Automated Image Analysis Reveals Different Localization of Synaptic Gephyrin C4 Splice Variants

“…High reads per kilobase million were detected for the 22 exons coding for the Gphn isoform P1. The expression of E2 and any of the recently discovered exons ( Dos Reis et al, 2022 ) was below the limit of detection in the dataset. The expression profile in cortical [glutamatergic; CamKII (sodium channel epithelial 1 subunit α; Scnn1a), Grik4; and GABAergic (SST, parvalbumin; Pvalb, vasointestinal peptide; Vip)] neurons was comparable to the cells in the hippocampus (Extended Data Fig.…”

“…The murine gephyrin gene ( Gphn ) was originally described to consist of 29 exons ( Heck et al, 1997 ; Meier et al, 2000 ; Ramming et al, 2000 ; Fritschy et al, 2008 ). A recent targeted approach, together with long-read sequencing, discovered 40 exons, and alternative splicing results in the expression of 277 unique transcripts ( Dos Reis et al, 2022 ). The major gephyrin isoform in the brain is referred to as P1 and is generated from 22 exons ( Prior et al, 1992 ; Fritschy et al, 2008 ; Dos Reis et al, 2022 ).…”

“…A recent targeted approach, together with long-read sequencing, discovered 40 exons, and alternative splicing results in the expression of 277 unique transcripts ( Dos Reis et al, 2022 ). The major gephyrin isoform in the brain is referred to as P1 and is generated from 22 exons ( Prior et al, 1992 ; Fritschy et al, 2008 ; Dos Reis et al, 2022 ). The P1 open reading frame of human GPHN is 94% homologous to the rodent one, which translates to 99.7% amino acid conservation ( Rees et al, 2003 ).…”

“…The P1 open reading frame of human GPHN is 94% homologous to the rodent one, which translates to 99.7% amino acid conservation ( Rees et al, 2003 ). All internal exons may undergo alternative splicing ( Dos Reis et al, 2022 ) and traditionally certain exons were named according to the resulting alternative sequences in either of the three protein domains, e.g., G2, C3, C4a, C4c, C4d, and E2 ( Fritschy et al, 2008 ; Fig. 1 a ).…”

Molybdenum Cofactor Deficiency in Humans

“…Vertebrate gephyrin is encoded by the GPHN gene, composed of up to 40 exons, with a varying number of exons undergoing alternative splicing [ 74 ]. Although the number of exons and alternative splice products reported in humans is far lower [ 75 ] than recently reported in mice, it is expected that the highly mosaic gene expression also holds true in humans and reflects the complex regulation of gephyrin function in the central nervous system.…”