Abstract
Cadherins and neuroligins (NLs) represent two families of cell adhesion proteins that are essential for the establishment of synaptic connections in vitro; however, it remains unclear whether these proteins act in concert to regulate synapse density. Using a combination of overexpression and knockdown analyses in primary hippocampal neurons, we demonstrate that NL1 and N-cadherin promote the formation of glutamatergic synapses through a common functional pathway. Analysis of the spatial relationship between N-cadherin and NL1 indicates that in 14-day in vitro cultures, almost half of glutamatergic synapses are associated with both proteins, whereas only a subset of these synapses are associated with N-cadherin or NL1 alone. This suggests that NL1 and N-cadherin are spatially distributed in a manner that enables cooperation at synapses. In young cultures, N-cadherin clustering and its association with synaptic markers precede the clustering of NL1. Overexpression of N-cadherin at this time point enhances NL1 clustering and increases synapse density. Although N-cadherin is not sufficient to enhance NL1 clustering and synapse density in more mature cultures, knockdown of N-cadherin at later time points significantly attenuates the density of NL1 clusters and synapses. N-cadherin overexpression can partially rescue synapse loss in NL1 knockdown cells, possibly due to the ability of N-cadherin to recruit NL2 to glutamatergic synapses in these cells. We demonstrate that cadherins and NLs can act in concert to regulate synapse formation.