N-cadherin and Neuroligins Cooperate to Regulate Synapse Formation in Hippocampal Cultures

Mytyl Aiga,Joshua N Levinson,Shernaz X Bamji

doi:10.1074/jbc.m110.176305

Mytyl Aiga, Joshua N Levinson + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m110.176305

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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.

Highlights

The distributions of cadherins and neuroligins (NLs)2 at synaptic compartments have previously been analyzed; their spatial distribution with respect to one another is still unclear
To examine the temporal relationship of N-cadherin and NL1 clustering at synapses, cells were immunolabeled with anti-VGlut-1 and anti-N-cadherin
Disruption of either cadherin or neurexin-neurolignin adhesion complexes in cultured hippocampal neurons has been shown to drastically reduce synapse density, suggesting key roles for these adhesive systems in synapse formation [5,6,7]. Both N-cadherin and NL1 have been shown to localize primarily to glutamatergic synapses, it was previously unclear whether they colocalize at individual synapses or whether they localize to different synaptic subtypes and if these adhesion systems function in concert to regulate synapse formation

Summary

To whom correspondence should be addressed

This information is essential for understanding the functional interplay between these two adhesion systems. Cadherins and their associated catenins have been observed in both pre- and postsynaptic compartments in many neuronal populations in the CNS [8, 9]. In stark contrast to what has been shown for cadherins, overexpression of NLs in cultured hippocampal neurons dramatically enhances the formation of glutamatergic and GABAergic synapses and increases spine number [20, 24, 25]. We demonstrate that approximately half of glutamatergic synapses express both adhesion proteins, indicating that these molecules are spatially distributed in such a way as to enable functional cooperation. Using knockdown and overexpression analyses, we demonstrate that NL1 and N-cadherin mediate synapse formation via a common pathway

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION