Abstract
N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO) cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of choline acetyltransferase (ChAT) transcriptional regulatory elements (ChATBACEGFP) were cultured as tissue explants for 5 days and cocultured with transfected CHO cells for an additional 2 days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing α3 and β4 nicotinic acetylcholine receptor (nAChR) subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs) and miniature EPSPs (mEPSPs). EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin (TTX). These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.
Highlights
Cell adhesion molecules localized at developing synapses mediate homo and heterotypic interactions between pre and postsynaptic membranes
To examine whether any other type I or type II cadherin was expressed in Chinese Hamster Ovary (CHO) cells, cultured CHO cells were immunolabeled with anti p120-catenin antibodies. p120-catenin binds to the membrane proximal region of the cytoplasmic domain of type I and type II cadherins, and no other cell membrane protein is known to bind p120-catenin (Reynolds et al, 1996; Anastasiadis and Reynolds, 2000)
In CHO cells transfected with enhanced green fluorescent protein (EGFP), p120catenin localized to the cytosol, while in cells transfected with N-cadherin-EGFP, the subcellular localization of p120-catenin shifted to the cell membrane (Figure 1B) (Rubio et al, 2005)
Summary
Cell adhesion molecules localized at developing synapses mediate homo and heterotypic interactions between pre and postsynaptic membranes. These trans-synaptic interactions can initiate synapse formation and promote synaptic maturation by providing adhesive support between opposing cell membranes, and by regulating intracellular signaling leading to the assembly of pre and postsynaptic machineries (Dalva et al, 2007; Benson and Huntley, 2011). N-cadherin trans-synaptically regulates transmitter release in cholinergic neurons through a mechanism modulated by SAP97 (Neff et al, 2009). This evidence indicates that N-cadherin homotypic interaction across the synaptic cleft
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