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

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.Synapse formation begins with the recognition of appropriate targets and formation of incipient contacts and is followed by the recruitment of pre-and postsynaptic proteins to exquisitely localized microdomains at points of cell-cell contact (1, 2). Transsynaptic cell adhesion complexes have come to the forefront as key players in the formation and maturation of synaptic connections (3, 4). Among these adhesion complexes, the homophilic cadherin complex and the heterophilic neurexin-neuroligin complex have been extensively studied and are known to exert key roles in synapse development (5-7).The distributions of cadherins and neuroligins (NLs) 2 at synaptic compartments have previously been analyzed; however, their spatial distribution with respect to one another is still unclear. 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). Previous reports demonstrate that in the brain, two of these, N-and E-cadherin, are localized to synaptic complexes in mutually exclusive distributions (8). As synapses mature, N-cadherin is excluded from inhibitory synapses, becoming primarily localized at glutamatergic synapses (10).Evidence that cadherins play an important role in establishing synaptic junctions includes observations that cadherins rapidly accumulate at points of cell-cell contact prior ...

N-Cadherin Prodomain Processing Regulates Synaptogenesis

“…Our results are in agreement with a previous report in which suppression of N-cad function by overexpression of a dominant-negative mutant was shown to impair presynaptic organization and synapse number (Togashi et al, 2002;Vitureira et al, 2012). Furthermore, Aiga et al (2011) have recently shown that synapse density increases following N-cad overexpression (Aiga et al, 2011) only in young neurons. The discrepancy between our study and those that show no effect on synapse number after N-cad overexpression (Okamura et al, 2004;Vitureira et al, 2012) could be attributed to the superior transfection rate (Ͼ60%) in the current study, Figure 8.…”

N-cadherin expression is regulated by UTP in schwannoma cells

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Rare autosomal copy number variations in early-onset familial Alzheimer’s disease

“…The 488-kb gain in CN encompasses CDH2, encoding N-cadherin, which is expressed in brain, skeletal and cardiac muscles and has critical roles in synaptic adhesion, dendritic morphology and neuritic growth. [55][56][57][58][59] CDH2 has been extensively studied in AD. For example, inhibition of N-cadherin function has been reported to accelerate Ab-triggered synapse damage.…”