Afadin regulates puncta adherentia junction formation and presynaptic differentiation in hippocampal neurons

PLoS One. 2014 Feb 27;9(2):e89763. doi: 10.1371/journal.pone.0089763. eCollection 2014.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism
  • Brain / pathology
  • Cadherins / metabolism
  • Cell Adhesion Molecules / genetics
  • Cell Adhesion Molecules / metabolism
  • Mice
  • Mice, Knockout
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism*
  • Mossy Fibers, Hippocampal / metabolism
  • Nectins
  • Presynaptic Terminals / metabolism
  • Pyramidal Cells / metabolism*
  • Synapses / metabolism*


  • Cadherins
  • Cell Adhesion Molecules
  • Microfilament Proteins
  • Nectin1 protein, mouse
  • Nectin3 protein, mouse
  • Nectins
  • afadin

Grant support

This work was supported by the Global COE Program “Global Center for Education and Research in Integrative Membrane Biology” ( and the Targeted Proteins Research Program ( from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, Grants-in-Aid for Scientific Research (S) (21227005 to Y.T.) and (C) (24590354 to K.M. and 22500293 to H.T.) and for Young Scientists (B) (25860214 to T.M.) from the Japan Society for the Promotion of Science (, and the grants (to Y.T.) from the Naito Foundation (, the Sagawa Foundation for Promotion of Cancer Research (, and the Yasuda Medical Foundation ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.