Delta-catenin regulates spine and synapse morphogenesis and function in hippocampal neurons during development

Abstract

The maintenance of spine and synapse number during development is critical for neuronal circuit formation and function. Here we show that delta-catenin, a component of the cadherin-catenin cell adhesion complex, regulates spine and synapse morphogenesis during development. Genetic ablation or acute knockdown of delta-catenin leads to increases in spine and synapse density, accompanied by a decrease in tetrodotoxin induced spine plasticity. Our results indicate that delta-catenin may mediate conversion of activity-dependent signals to morphological spine plasticity. The functional role of delta-catenin in regulating spine density does not require binding to cadherins, but does require interactions with PDZ domain-containing proteins. We propose that the perturbations in spine and synaptic structure and function observed after depletion of delta-catenin during development may contribute to functional alterations in neural circuitry, the cognitive deficits observed in mutant mice, and the mental retardation pathology of Cri-du-chat syndrome.

Figure 1.

Reduction in the levels of N-cadherin and pan-cadherin in hippocampal lysates from δ-catenin +/+ and δ-catenin −/− mice. Western blot analysis of hippocampal lysates from P18 δ-catenin +/+ and δ-catenin −/− mice with antibodies to N-cadherin, pan-cadherin, p120ctn, αN-catenin, β-catenin, and β-tubulin.

Figure 2.

Characteristics of spines from hippocampal neurons from δ-catenin +/+ and δ-catenin −/− mice (DIV 17). Representative images of spines from δ-catenin +/+ and δ-catenin −/− hippocampal neurons. Scale bar, 5 μm. δ-cat, δ-Caternin.

Figure 3.

Hippocampal neurons from δ-catenin −/− mice have increased excitatory but not inhibitory synaptic function. A, Representative traces of mEPSCs from δ-catenin +/+ and δ-catenin −/− neurons. B, Representative traces of mIPSCs from δ-catenin +/+ and δ-catenin −/− neurons. δ-cat, δ-Catenin.

Figure 4.

Acute shRNA-mediated knockdown of δ-catenin leads to an increase in spine density. Representative images of spines from neurons transfected with the vector or shRNA to δ-catenin at DIV 11 and examined at DIV 17. Scale bar, 5 μm.

Figure 5.

Acute shRNA-mediated knockdown of δ-catenin is accompanied by an increase in excitatory synaptic density and function. A, Representative images from rat hippocampal neurons transfected with vector or shRNA to δ-catenin at DIV 11 and examined at DIV 17, immunostained with an antibody to PSD95, an excitatory postsynaptic marker. B, Representative images from rat hippocampal neurons transfected with vector or shRNA to δ-catenin at DIV 11 and examined at DIV 17, immunostained with an antibody to vGlut1, an excitatory presynaptic marker. Scale bar, 2μm. δ-cat, δ-Catenin. C, Representative traces of mEPSCs from neurons expressing vector, shRNA to δ-catenin, or shRNA to δ-catenin and a full-length shRNA-resistant construct of δ-catenin. D, Frequency of mEPSCS in neurons expressing vector, shRNA to δ-catenin, or shRNA to δ-catenin and a full-length shRNA-resistant construct of δ-catenin. E, Amplitude of mEPSCS in neurons expressing vector, shRNA to δ-catenin, or shRNA to δ-catenin and a full-length shRNA-resistant construct of δ-catenin (p < 0.05).

Figure 6.

Acute δ-catenin knockdown-mediated increase in spine density is independent of binding of cadherin. A, Schematic of constructs of full-length δ-catenin and Δ-arm δ-catenin. B, Representative images of spines from rat hippocampal neurons transfected with vector, δ-catenin shRNA, δ-catenin shRNA plus δ-catenin Δ-arm, or δ-catenin shRNA plus δ-catenin* at DIV 11 and examined at DIV 17. Scale bar, 2 μm. C, Quantitation of spine density (per 10 μm) from rat hippocampal neurons expressing vector, shRNA, shRNA plus δ-catenin Δ-arm, or shRNA plus δ-catenin* (***p < 0.006). δ-cat, δ-Catenin.

Figure 7.

Regulation of spine density by δ-catenin requires the PDZ binding domain. A, Schematic of construct of δ-catenin N terminus, N-Arm, δ PDZ*, and full-length δ-catenin*. B, Representative images of spines from rat hippocampal neurons transfected with vector, δ-catenin shRNA, δ-catenin shRNA plus δ-catenin δ PDZ*, or δ-catenin shRNA plus δ-catenin* at DIV 11 and examined at DIV 17. Scale bar, 2 μm. C, Quantitation of spine density (per 10 μm) from rat hippocampal neurons expressing vector, shRNA, shRNA plus N terminus, shRNA plus N-Arm, shRNA plus δ-catenin δ PDZ*, or shRNA plus δ-catenin* (***p < 0.0001). δ-cat, δ-Catenin.

Figure 8.

Acute knockdown of δ-catenin inhibits TTX-mediated alterations in spine length. A, Representative images of spines from hippocampal neurons transfected with vector or δ-catenin shRNA and untreated or treated with TTX. Scale bar, 2 μm. B, Quantitation of spine length from rat hippocampal neurons transfected with vector or δ-catenin shRNA and untreated or treated with TTX (***p < 0.0001). δ-cat, δ-Catenin.