Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses

Abstract

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.

Roles of afadin in functional differentiations of hippocampal mossy fiber synapse

“…We have previously shown that the area of PSDs per unit volume of a spine is decreased and that the concentration of PSD‐95 is slightly decreased in the afadin ‐deficient hippocampal mossy fiber synapse (Sai et al . ). Although the exact mechanism for the formation of the incomplete PSDs in the afadin ‐deficient synapse is not known, these structural defects of the PSDs may at least partly explain the impairment of the postsynaptic functions in the afadin ‐deficient cultured hippocampal neurons.…”

“…Although the detailed role of afadin in this process remains elusive, it may facilitate the docking, priming, endocytosis, and/or recycling processes. The fine structures of the AZs are not sufficiently observed by serial block face‐scanning electron microscopy, but as the immunofluorescence signal for the AZ protein bassoon and the number of the docked SVs are decreased (Sai et al …”

Postnatal nectin‐3 knockdown induces structural abnormalities of hippocampal principal neurons and memory deficits in adult mice

“…Nectins bind with the adaptor protein, afadin, to form the nectin–afadin complex, which further cooperates with the N‐cadherin‐catenin complex in the puncta adherentia junctions (PAJs) (Mizoguchi et al, ; Tachibana et al, ; Togashi et al, ). Using the afadin ‐deficient mouse model, studies have shown that afadin deletion disrupts PAJs, reduces spine density, and alters the ultrastructural morphogenesis in the hippocampus (Beaudoin et al, ; Majima et al, ; Sai et al, ). Afadin deletion also induces nectin mislocation in the DG (Majima et al, ), without affecting nectin expression levels, suggesting that afadin may serve as a downstream molecule of nectins (Beaudoin et al, ).…”

NGL‐3‐induced presynaptic differentiation of hippocampal neurons in an afadin‐dependent, nectin‐1‐independent manner

“…We have further shown by serial block face‐scanning electron microscopy that the complexity of the postsynaptic spines and the mossy fiber boutons, the number of the spine heads, the area of the PSDs, and the density of the SVs docked to the AZs are decreased in the afadin ‐deficient synapse (Sai et al . ). Subsequently, using the same afadin ‐deficient mouse line, we have investigated the roles of afadin in the functional differentiation of the hippocampal mossy fiber synapse and shown electrophysiologically that both the release probability of glutamate and the postsynaptic responsiveness to glutamate are markedly reduced in the afadin ‐deficient mossy fiber synapse, whereas neither long‐term potentiation nor long‐term depression is affected (Geng et al .…”

“…Afadin plays a role in the formation of the PAJs by first binding to both presynaptic nectin‐1 and postsynaptic nectin‐3 and then binding to αN‐catenin, eventually recruiting N‐cadherin via its direct binding protein β‐catenin to the adhesion sites formed by the trans ‐interaction of nectin‐1 and nectin‐3 (Sai et al . ). In contrast, the modes of action of afadin in other differentiation processes of the mossy fiber synapse have not been investigated.…”