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

Postsynaptic scaffolding proteins function as central organization hubs, ensuring the synaptic localization of neurotransmitter receptors, trans-synaptic adhesion proteins, and signaling molecules. Gephyrin is the major postsynaptic scaffolding protein at glycinergic and a subset of GABAergic inhibitory synapses. In contrast to cells outside the CNS, where one gephyrin isoform is predominantly expressed, neurons express different splice variants. In this study, we characterized the expression and scaffolding of neuronal gephyrin isoforms differing in the inclusion of the C4 cassettes located in the central C-domain. In hippocampal and cortical neuronal populations, gephyrin P1, lacking additional cassettes, is the most abundantly expressed isoform. In addition, alternative splicing generated isoforms carrying predominantly C4a, and minor amounts of C4c or C4d cassettes. We detected no striking difference in C4 isoform expression between different neuron types and a single neuron can likely express all C4 isoforms. To avoid the cytosolic aggregates that are commonly observed on exogenous gephyrin expression, we used adeno-associated virus (AAV)-mediated expression to analyze the scaffolding behavior of individual C4 isoforms in murine dissociated hippocampal glutamatergic neurons. While all isoforms showed similar clustering at GABAergic synapses, a thorough quantitative analysis revealed localization differences for the C4c isoform (also known as P2). Specifically, synaptic C4c isoform clusters showed a more distal dendritic localization and reduced occurrence at P1-predominating synapses. Additionally, inhibitory currents displayed faster decay kinetics in the presence of gephyrin C4c compared with P1. Therefore, inhibitory synapse heterogeneity may be influenced, at least in part, by mechanisms relating to C4 cassette splicing.Significance StatementA neuron receives and integrates thousands of different synaptic inputs. At synapses of the same type, i.e., using the same neurotransmitter, subtle differences in their molecular composition could account for functional changes. Diverse synapse populations could contain different isoforms of the same protein, which are themselves generated by alternative splicing. In this work, we focused on the neuronal isoforms of gephyrin, which organizes structures at most inhibitory synapses. While overall similar properties of all isoforms were observed, an in-depth automated analysis revealed that the localization of clusters from one of the isoforms was different. Finally, this indicates that the alternative splicing of gephyrin could be one of the factors influencing the organization of postsynaptic proteins at diverse synapse populations.