α5 Integrin Signaling Regulates the Formation of Spines and Synapses in Hippocampal Neurons

Huaye Zhang,Donna J. Webb,Alan F. Horwitz,Devi Majumdar

doi:10.1074/jbc.m610981200

Huaye Zhang, Donna J. Webb + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m610981200

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Abstract

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.

Highlights

Integrins are heterodimeric, transmembrane cell surface receptors that mediate cell-cell and cell-matrix interactions
Expression of constitutively active or dominant negative ␣5 integrin resulted in alterations in the number of dendritic protrusions, spines, and synapses. ␣5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1
Expression of human ␣5 rescued the defects in the small interfering RNA (siRNA)-expressing neurons (Fig. 1D). These results show that the defects in spine morphogenesis and synapse formation are due to the loss of endogenous ␣5 integrin and point to a critical function for this integrin in the regulation of spine and synapse formation

Summary

Integrin Signaling Regulates Spine and Synapse Formation

The GIT1 siRNA has been previously described [15]. Human ␣5 integrin-GFP was prepared as previously described [16]. SiRNA-expressing neurons are very smooth with a decrease in the number of spines compared with pSUPER (Control)-transfected cultures. The ␣5 siRNA-expressing neuron had significantly fewer synapses, as determined by immunostaining with the synaptic marker SV2. D, quantification of the number of spines, protrusions, and synapses in neurons transfected with ␣5 siRNA or pSUPER empty vector (Control). The defects of ␣5 siRNA on spine morphogenesis and synapse formation were reversed by expression of human ␣5-GFP (Rescue). Were transfected by a modified calcium phosphate method as previously described [20]. Nonspecific absorption was blocked by incubating the coverslips with 20% goat serum in phosphate-buffered saline for 1 h at room temperature.

EXPERIMENTAL PROCEDURES

RESULTS

Expression of the Signaling