By specifically deleting afadin
in hippocampal excitatory neurons before synapse formation, we were able to examine the role of this scaffold protein in regulating neuronal differentiation in the presence of normal glia and other cells. Much of the differentiation of mutant hippocampal CA1 pyramidal neurons proceeded comparatively normally. A few neuronal cell bodies were mislocalized, but most were localized appropriately in the stratum pyramidale. Similarly, dendrite growth and branching appeared comparatively normal in the mutant neurons. Three significant phenotypes were observed in the mutant neurons. Spine formation was impaired because only 60% the normal density was observed in the mutant CA1 stratum radiatum. A similar reduction in density of excitatory synapses in the stratum radiatum was also observed. Finally, the mutants exhibited a dramatic loss of 70% of the N-cadherin puncta and similar reductions in the densities of β-catenin and αN-catenin puncta with no changes in total expression of either N-cadherin or these catenins, documenting afadin's role in cadherin recruitment. It seems likely that many of the residual cadherin and catenin puncta are expressed in cells not targeted by Nex-Cre
. Notably, these synaptic deficits appear much more dramatic than that observed following targeted deletion of N-cadherin (Kadowaki et al., 2007
). Those authors observed the continued presence of β-catenin at synapses, indicating that other classical cadherins remained at these synapses. Prior work has suggested that afadin promotes the recruitment and activation of all of the classical cadherins (Takai et al., 2008
). Interestingly, we did not observe effects of afadin deletion on the density or properties of Nectin-1 and Nectin-3 puncta or on total expression levels of either nectin, consistent with the possibility that these proteins function upstream of afadin in controlling cadherin and catenin localization and function. In contrast, absence of afadin does result in mislocalization of nectins in the dentate gyrus and intestinal epithelium (Majima et al., 2009
; Tanaka-Okamoto et al., 2011
). The reason for this discrepancy is not clear, but suggests that there are cell type-specific mechanisms that control localization of the nectins, possibly involving other scaffold proteins, such as S-SCAM (Yamada et al., 2003
). Overall, our data is consistent with the possibility that afadin promotes synapse formation/maintenance through recruitment of cadherins and catenins, but does not significantly perturb pre- or post-synaptic function.
In synaptic structure analyses, both light level immunofluorescence (not shown) and EM analysis detected a small but significant increase in the size of presynaptic nerve terminals in mutant compared to control CA3 neurons. Analyses of presynaptic function—probability of release and sizes of the readily-releasable and reserve vesicle pools—indicates that the mutant CA3 presynaptic nerve terminals functioned normally. Additionally, absence of afadin did not affect the average amount of bassoon associated with each synapse as determined by STORM. Similarly, postsynaptic morphology as assessed by appearance and length of the post-synaptic density appeared normal as assessed by EM and AMPA receptor GluR1/2 content at these synapses was normal as assessed by STORM. Quantification of CA3 fiber volley amplitude indicated that normal numbers of mutant CA3 pyramidal cell axons invaded the CA1 stratum radiatum. Postsynaptic responses were reduced by approximately the same percentage as the reductions in synapse density quantified by EM and STORM. Mutant synapses had similar number of glutamate receptors juxtaposed to bassoon. Perhaps, the increase in bouton size is an indirect result of fewer synapses and/or spines. Taken together, these analyses indicate that the synapses formed in the absence of afadin function normally.
These findings contrast with previous mouse genetic models for cadherin complex function, but comparisons are complicated by the presence of several functional homologues for the cadherins and catenins. Animals lacking N-cadherin clearly form CNS synapses in vitro
, but synaptic density has not been quantified in this mutant in vivo
and other cadherins almost certainly remain at these synapses (Kadowaki et al., 2007
). Similarly, absence of αN-catenin does not prevent synapse formation in vivo
although, similar to cadherin inhibition, this results in spine and synapse instability in culture (Abe et al., 2004
; Park et al., 2002
; Togashi et al., 2002
). Absence of β-catenin results in a small increase in CA1 synapse density with a small, but significant deficit in the synaptic vesicle reserve pool (Bamji et al., 2003
). Possible compensation of the αN-catenin and β-catenin mutants by other α-catenin family members and the β-catenin homologue plakoglobin were not examined in these studies. Mice with a targeted deletion of p120-catenin had reduced numbers of dendritic spines and synapses in CA1-SR, and a reduction in N-cadherin expression (Elia et al., 2006
; Takeichi, 2007
). In vitro
analysis suggested that the loss of spines in this mutant is attributable to mis-regulation of Rac and Rho, while normal maturation of spines does require interactions of cadherins with p120-catenin (Elia et al., 2006
; Ishiyama et al., 2010
). In contrast, deletion of the p120-catenin homologue, δ-catenin results in deficits in synaptic transmission and plasticity without detectable alterations of synapse density (Israely et al., 2004
). Importantly, absence of this gene also results in premature loss of synaptic transmission, synapses, spines and dendrites in the adult cortex (Matter et al., 2009
). Considering these phenotypes, that of the p120-catenin
mutant appears most similar to that of the afadin
mutant, consistent with the role of p120-catenin in mediating an interaction between afadin and cadherins.
Through its PDZ domain, afadin also binds at least three other families of cell surface receptors, the nectins, EphB receptors and neurexins (Beaudoin, 2006
) Interestingly, mice with triple mutants of neuroligin or α-neurexin have profoundly impaired synaptic transmission with only small alterations in numbers of inhibitory and excitatory synapses (Missler et al., 2003
; Varoqueaux et al., 2006
). In contrast, the triple EphB1/B2/B3
null mice exhibit an approximately 50% decrease in the density of spines in CA1 with a reduced number of spine and increased number of shaft synapses as well as striking reductions in spine maturation and post-synaptic compartment maturation (Henkemeyer et al., 2003
). Thus, absence of afadin may impair EphB receptor function, as we found a reduction in EphB2 puncta density, but these receptors clearly have additional functions since the triple EphB receptor
mutant has phenotypes not observed in the afadin
mutant. In contrast to mutants lacking EphB receptors, neuroligins or α-neurexins, afadin appears to promote synapse density without having major effects on synapse structure or function.
This study focused on afadin because it lacks close structural homologues, thereby preventing compensation by other proteins. Nonetheless, a significant number of synapses form in its absence. As a possible explanation, a recent report raises the possibility that the PDZ-domain containing protein S-SCAM acts as a functional homologue of afadin through mediating association of N-cadherin with Neuroligin-1 (Stan et al., 2010
). Additionally, recent studies also suggest that N-cadherin function can be promoted through extracellular interactions with nectin-2 and protocadherin-19 (Biswas et al., 2010
; Morita et al., 2010
). It is not clear whether the resulting stimulation of N-cadherin function depends upon afadin, p120-catenin or other members of the cadherin complex. Thus, other cytoplasmic scaffold and surface proteins may promote cadherin function in the absence of afadin.
Additional interactions of afadin may also promote synapse formation. For example its RA domain binds Rap1 (Takai et al., 2008
). Previous studies in cortical cultures have provided evidence that afadin may function in a pathway through which NMDA receptors control spine maturation and AMPA receptor recruitment through Rap1 (Xie et al., 2005
). Additionally, afadin may promote N-cadherin-dependent spine growth and Rac activation by recruiting the Rac-GEF, kalirin-7 (Xie et al., 2008
). However, as loss of kalirin-7 causes no hippocampal spine or synaptic abnormalities, it is unlikely to underlie the defects seen in the afadin
null hippocampus seen in our study (Cahill et al., 2009
; Xie et al., 2011
). Though as afadin interacts with N-cadherin and p120-catenin, misregulation or mislocalization of Rho family GTPases may still contribute to the reduction in spine density observed in the mutant in vivo
In conclusion our data provide strong evidence that absence of afadin results in significant reductions in cadherin puncta, spine and excitatory synapse density in the CA1-SR without affecting function of those synapses, which are formed in its absence. The data suggest that impaired clustering and activity of cadherins contributes to the phenotypes observed following neuron-specific deletion of afadin. The results do not exclude possible additional contributions to the afadin mutant phenotype from mis-regulation of neurexin, EphB or nectin activity within the brain.