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. 2007 Aug 14;104(33):13479-84.
doi: 10.1073/pnas.0702334104. Epub 2007 Aug 6.

beta-Catenin regulates excitatory postsynaptic strength at hippocampal synapses

Takashi Okuda  1 , Lily M Y Yu, Lorenzo A Cingolani, Rolf Kemler, Yukiko Goda
Affiliations

beta-Catenin regulates excitatory postsynaptic strength at hippocampal synapses

Takashi Okuda et al. Proc Natl Acad Sci U S A. .

Abstract

The precise contribution of the cadherin-beta-catenin synapse adhesion complex in the functional and structural changes associated with the pre- and postsynaptic terminals remains unclear. Here we report a requirement for endogenous beta-catenin in regulating synaptic strength and dendritic spine morphology in cultured hippocampal pyramidal neurons. Ablating beta-catenin after the initiation of synaptogenesis in the postsynaptic neuron reduces the amplitude of spontaneous excitatory synaptic responses without a concurrent change in their frequency and synapse density. The normal glutamatergic synaptic response is maintained by postsynaptic beta-catenin in a cadherin-dependent manner and requires the C-terminal PDZ-binding motif of beta-catenin but not the link to the actin cytoskeleton. In addition, ablating beta-catenin in postsynaptic neurons accompanies a block of bidirectional quantal scaling of glutamatergic responses induced by chronic activity manipulation. In older cultures at a time when neurons have abundant dendritic spines, neurons ablated for beta-catenin show thin, elongated spines and reduced proportion of mushroom spines without a change in spine density. Collectively, these findings suggest that the cadherin-beta-catenin complex is an integral component of synaptic strength regulation and plays a basic role in coupling synapse function and spine morphology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Synapse morphology in neurons ablated for postsynaptic β-catenin. (A) Dissociated cells from homozygous β-catenin-floxed mice were transfected with Cre-IRES-GFP and grown in culture. Cre-expressing neuron, identified by the GFP fluorescence (green), shows reduced β-catenin expression compared with a neighboring nontransfected neuron (red) at DIV7. (B) Representative synapsin I localization on dendrites of neurons transfected with control GFP or Cre-IRES-GFP. Postsynaptic β-catenin loss has no significant effect on synapse density (see Results). (C) Representative GFP-actin expression in dendrite segments of DIV18 β-catenin-floxed neurons. Coexpression of Cre produces elongated spines compared with a control cell. [Scale bars: 40 μm (A), 5 μm (B and C).] (D Left) Relative proportion of mushroom-shaped (gray bars) vs. filopodial (open bars) spines in control (n = 9) and Cre-positive (n = 13) neurons (P < 0.05). (Right) Comparison of mean spine density along 10-μm dendritic length between control (n = 9) and Cre (n = 13)-transfected neurons (P > 0.2).
Fig. 2.
Fig. 2.
Effects of β-catenin loss on glutamatergic quantal responses. (A) Representative traces of mEPSCs from β-catenin-floxed neurons expressing GFP alone or Cre at DIV14. (Scale bars: 100 ms, 20 pA.) (B) Cumulative distribution plots of the mean mEPSC amplitude from GFP- (solid line) or Cre-expressing neurons (dashed line) (P < 0.05; n = 15 cells each). (C) Examples of average mEPSC trace from GFP- (solid line) or Cre-expressing neurons (dotted line). Traces on the right have been scaled to the peak. (Scale bars: 5 ms, 5 pA.) (D Left) Summary of mean mEPSC amplitude. Coexpression of WT β-catenin with Cre rescued the mean mEPSC amplitude to control levels (n = 8; P > 0.4 vs. control), but not by ΔARM (n = 9; P > 0.6 vs. Cre). (Right) Summary of mean mEPSC frequency. The mean mEPSC frequency is unaltered in Cre-expressing neurons compared with controls (P > 0.5; n = 15 cells each).
Fig. 3.
Fig. 3.
DN N-cadherin mimics the effect of β-catenin loss. (A) Illustration of mutant N-cadherin constructs used. (B) Cell dissociation assay in HEK293 cells, comparing the efficacy of N-cadherin mutants for impairing Ca2+-dependent cell adhesion relative to control. WT, P > 0.2; DN-N-cad, P < 0.01; N-cadΔC, P > 0.7; n = 4 each. Cell aggregation index was defined as the percent ratio of [NTE − NTC]/NTE, where NTC and NTE were the cell particle number after the TC and TE treatments, respectively. (C) The mean mEPSC amplitudes from neurons expressing GFP alone (control, n = 15) compared with WT N-cadherin (WT, P > 0.3, n = 12), the DN N-cadherin (DN, P < 0.01, n = 14), or N-cadherinΔC (ΔC, P > 0.1, n = 10).
Fig. 4.
Fig. 4.
The PDZ-binding motif of β-catenin is important for regulating mEPSC size. (A) Schematic diagram of β-catenin deletion mutants. (B) Interaction of β-catenin deletion mutants with N-cadherin or αN-catenin in HEK293 cells. Western blots show coimmunoprecipitation (IP) of endogenous N-cadherin (Middle) and exogenous αN-catenin-HA (Bottom) with exogenously expressed Myc-tagged β-catenin mutants (Top). (C) Summary of mean mEPSC size in WT cultures overexpressing β-catenin constructs. Compared with control GFP neurons (n = 12), expression of WT (n = 10), ΔN (n = 10), or L132A (n = 10) had no effect (P > 0.7), whereas expression of ΔARM (n = 11), ΔC (n = 12), and ΔPDZ (n = 10) mutants reduced mEPSC amplitude (P < 0.05). (D) Summary of mean mEPSC amplitude in β-catenin-floxed neurons. The decreased mean mEPSC amplitude upon loss of β-catenin (P < 0.05; control, n = 9; Cre, n = 9; also see Fig. 2C) was rescued by coexpression of WT β-catenin (P > 0.1 vs. control; n = 7) or L132A (P > 0.7 vs. control; n = 9), but not by ΔPDZ (P < 0.01 vs. control n = 10) in parallel experiments.
Fig. 5.
Fig. 5.
Activity-dependent synaptic scaling is impaired upon β-catenin loss. (A) Cumulative distribution plots of the mean mEPSC amplitudes from untransfected control (Upper) or Cre-transfected neurons (Lower) after culturing with no drug (solid line), TTX (dotted line), or bicuculline (dashed line) for 2 days. (B) Summary of the mean mEPSC amplitudes after chronic activity manipulations. In control neurons, relative to no drug treatment (n = 16), mEPSC size was scaled up in TTX (P < 0.01; n = 15) or down in bicuculline (P < 0.01; n = 12). In β-catenin-null neurons, relative to no drug treatment (n = 16), neither chronic TTX (P > 0.6; n = 16) nor bicuculline (P > 0.1; n = 13) caused a significant change in the mean mEPSC size.
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