Elsevier

Neuropharmacology

Volume 47, Issue 5, October 2004, Pages 779-786
Neuropharmacology

Multiple functions for the cadherin/catenin complex during neuronal development

https://doi.org/10.1016/j.neuropharm.2004.07.031Get rights and content

Abstract

The cadherin/catenin complex is a major transmembrane signaling complex through which extracellular signals can influence the cytoskeleton. It is present in neuronal processes during early development (J. Neurosci. 18 (1998) 6892) and appears to play multiple roles during neural circuit formation and maturation (Nat. Neurosci. 7 (2004) 357; Neuron 35 (2002) 77; Nat. Neurosci. 6 (2003) 1169). In previous work (Nat. Neurosci. 6 (2003) 1169), we showed that molecular manipulation of the cadherin/catenin complex has significant effects on the dendritic aborization of cultured hippocampal neurons at 9 days in vitro (d.i.v.). Here we extend these observations by examining the effects of over-expression of β-catenin (fused to GFP; GFP-β-cat) or sequestering endogenous β-catenin [using the intracellular domain of N-cadherin; Ncad(intra)] in cultured hippocampal neurons at different developmental stages. When transfected at 1 d.i.v. and assayed 48 h later, over-expression of GFP-β-cat increased axonal length and complexity, while sequestering endogenous β-catenin had the opposite effect. Dendritic aborization was also enhanced by GFP-β-cat expression throughout early development although the magnitude of this effect decreased as neurons matured from 3 to 12 d.i.v. Finally, at 12 d.i.v., GFP-β-cat expression increased the density of dendritic spines although not the relative proportion of different subtypes of spines. These results demonstrate that the cadherin/catenin complex plays multiple roles in the development of neuronal morphology and that these roles change as neurons mature.

Introduction

Early neuronal development consists of a stereotypic progression of events beginning with neurite extension, differentiation of the axon, dendritic arborization and synapse formation. This multi-step process involves many signaling molecules that mediate interactions between the cytoskeleton and the extracellular environment (Cline, 2001, Luo, 2002, McAllister, 2000, Scott and Luo, 2001, Wong and Ghosh, 2002), among them the cadherin/catenin complex (Takai et al., 2003, Tepass et al., 2000, Yap et al., 1997). Classical cadherins form homophilic interactions with cadherins on neighboring cells through their extracellular domains and intracellularly bind to β-catenin, which in turn associates with α-catenin and the actin cytoskeleton (Yap et al., 1997). Cadherins and catenins are present in both axons and dendrites early in development (Benson and Tanaka, 1998) and concentrate at axo-dendritic contacts upon initial synaptic formation, colocalizing with synaptic proteins (Togashi et al., 2002). Cadherins and catenins are also present in mature spines and shaft synapses, again colocalizing with synaptic proteins (Togashi et al., 2002).

In previous work, we showed that the cadherin/catenin complex plays a critical role in regulating dendritic morphogenesis (Yu and Malenka, 2003). Over-expression of any member of the cadherin/catenin complex was sufficient to enhance dendritic arborization at 9 d.i.v., while interfering with the function of the cadherin/catenin complex by over-expressing the intracellular domain of N-cadherin [Ncad(intra)] reduced the number of dendritic branches. We further showed that this effect is primarily, if not exclusively, due to increased cell adhesion via the cadherin/catenin complex and increased stabilization of the actin cytoskeleton in neuronal processes, and not transcriptional activation via nuclear β-catenin and LEF/TCF proteins. Here we extend our study by investigating the role of the cadherin/catenin complex at multiples stages of neuronal development. We show that early in development, at 3 d.i.v., in addition to enhancing dendritic arborization, over-expression of β-catenin also accelerates the growth and arborization of axons. Conversely, over-expression of Ncad(intra) decreases axonal growth and arborization. In more mature neurons, at 12 d.i.v., β-catenin over-expression has a moderate effect on dendritic arborization and also increases the density of dendritic spines, while sequestering β-catenin decreases dendritic branch tip number and does not affect spine density.

Section snippets

Hippocampal cell culture and transfection

Dissociated hippocampal neuronal cultures were prepared from postnatal day 0 (P0) Sprague–Dawley rat pups as previously described (Yu and Malenka, 2003), according to the National Institute of Health guide for the care and use of laboratory animals. Neurons were plated at a density of 37,500–50,000 cells/cm2 on 12 mm coverslips coated with matrigel and transfected using calcium phosphate (Xia et al., 1996). Neurons transfected at 1 d.i.v., were plated on a monolayer of type I astrocytes

The cadherin/catenin complex regulates axonal outgrowth and arborization

To examine the role of the cadherin/catenin complex in early axonal growth and arborization, we transiently expressed a stabilized form of β-catenin fused to the green fluorescent protein (GFP-β-cat) in cultured hippocampal neurons at 1 d.i.v. and analyzed the resulting phenotype at 3 d.i.v. Compared to cells transfected with GFP, neurons expressing GFP-β-cat had longer axons (Fig. 1A, B; 1.739±0.209, n=28, p<0.01 compared to 1±0.13, n=22) and more complex axonal branches as measured by the

Discussion

The cadherin/catenin complex is a transmembrane cell adhesion complex that is expressed at high levels in both axons and dendrites from early neuronal development (Benson and Tanaka, 1998) and provides a direct link to the actin cytoskeleton (Yap et al., 1997). In this study, we demonstrate that the cadherin/catenin complex can influence multiple stages of neuronal development, namely axonal growth and arborization, dendritic growth and arborization and dendritic spine formation. In neurons

Acknowledgements

This work was supported by a grant from the N.I.H. (MH63394).

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