Afadin Regulates Puncta Adherentia Junction Formation and Presynaptic Differentiation in Hippocampal Neurons

Daisaku Toyoshima,Hideru Togashi,Tomohiko Maruo,Yoshimi Takai,Masahiro Mori,Takahito Inoue,Kenji Mandai,Irwan Supriyanto

doi:10.1371/journal.pone.0089763

Abstract

The formation and remodeling of mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus are implicated in the cellular basis of learning and memory. Afadin and its binding cell adhesion molecules, nectin-1 and nectin-3, together with N-cadherin, are concentrated at puncta adherentia junctions (PAJs) in these synapses. Here, we investigated the roles of afadin in PAJ formation and presynaptic differentiation in mossy fiber-CA3 pyramidal cell synapses. At these synapses in the mice in which the afadin gene was conditionally inactivated before synaptogenesis by using nestin-Cre mice, the immunofluorescence signals for the PAJ components, nectin-1, nectin-3 and N-cadherin, disappeared almost completely, while those for the presynaptic components, VGLUT1 and bassoon, were markedly decreased. In addition, these signals were significantly decreased in cultured afadin-deficient hippocampal neurons. Furthermore, the interevent interval of miniature excitatory postsynaptic currents was prolonged in the cultured afadin-deficient hippocampal neurons compared with control neurons, indicating that presynaptic functions were suppressed or a number of synapse was reduced in the afadin-deficient neurons. Analyses of presynaptic vesicle recycling and paired recordings revealed that the cultured afadin-deficient neurons showed impaired presynaptic functions. These results indicate that afadin regulates both PAJ formation and presynaptic differentiation in most mossy fiber-CA3 pyramidal cell synapses, while in a considerable population of these neurons, afadin regulates only PAJ formation but not presynaptic differentiation.

Highlights

Synapses are specialized intercellular junctions that are indispensable for neuronal transmission
We examined the role of afadin in the accumulation of nectin-1, nectin-3 and N-cadherin at mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus
In the afadin-deficient neurons, the signals for nectin-1, nectin-3, N-cadherin and b-catenin decreased significantly. These results indicate that afadin is required for the accumulation of the immunofluorescence signals for the cell adhesion molecules (CAMs) and b-catenin at the synapses in cultured hippocampal neurons

Summary

Introduction

Synapses are specialized intercellular junctions that are indispensable for neuronal transmission. PAJs, in contrast, contain symmetrical paramembranous dense materials and are not associated with synaptic vesicles Both SJs and PAJs are highly developed as separate clusters consisting of distinctive macromolecular complexes in mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus. The synapses in this area undergo activity-dependent remodeling and reorganization, which is implicated in the cellular basis of learning and memory [2]. The molecular mechanisms underlying activity-dependent remodeling and reorganization are poorly understood It is not fully understood how contacts between axons and dendrites are initiated, or how presynaptic and postsynaptic components are recruited to the contact sites to establish synapses

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Conclusion