Volume 30 Issue 8 - August 5, 2016 PDF
The phospholipid-binding protein SESTD1 negatively regulates dendritic spine density by interfering with Rac1-Trio8 signaling pathway
Cheng-Che Lee, Chiung-Chun Huang and Kuei-Sen Hsu*
Department of Pharmacology, College of Medicine, National Cheng Kung University
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Dendritic spines are actin-rich protrusions from neuronal dendrites that harbor the majority of excitatory synapses. The balance of spine formation and retraction may influence dendritic integrity. While knowledge of the molecular mechanisms that promote dendritic spine formation has accumulated, little is known about the factors that limit spine formation. We show here that SESTD1, a phospholipid-binding protein containing a lipid-binding SEC14-like domain and two spectrin-repeat cytoskeleton interaction domains, negatively regulates dendritic spine density in cultured hippocampal neurons. Overexpression of SESTD1 decreases dendritic spine density in neurons by interfering with the interaction between Rac1 and its guanine nucleotide exchange factor (GEF) Trio8. Conversely, knockdown of SESTD1 increases dendritic spine density.  Further analysis reveals that the SPEC1 domain-mediated interaction with Rac1 is required for SESTD1 activity toward a decrease in dendritic spine density. Transfection of GEF domain of Trio8 into neurons rescues SESTD1-mediated decrease in dendritic spine density. More importantly, overexpression of SESTD1 results in a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs), whereas SESTD1 knockdown increases the mEPSC frequency. These results suggest that SESTD1 may act as a negative regulator of the Rac1-Trio8 signaling pathway to reduce dendritic spine density and lower excitatory synaptic transmission in hippocampal neurons.
Figure 1. SESTD1 negatively regulates dendritic spine and filopodia density in hippocampal neurons. (a) Representative images of hippocampal neurons co-transduced with EGFP-β-actin plus pEGFP-C1 vector or GFP-SESTD1. The bar graphs show the quantification of density of dendritic protrusion (spine and filopodia), spine, and filopodia (number/30 μm) of the representative groups. (b) Representative dendritic segments and quantification analysis showing the density of mature (PSD-95-positive) spine in UXIE vector- or SESTD1 (bicistronic expression plasmid)-transduced neurons at 17 DIV. (c) Effective and specificity of SESTD1-shRNAs. Western blotting showing total lysates of hippocampal neurons transduced with pLVTHM vector, shRNA constructs targeting SESTD1 (sh-SESTD1-I and sh-SESTD1-II) and control shRNA targeting DsRed (sh-DsRed) at 17 DIV using antibodies against SESTD1 and β-actin, respectively.
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