Dynamics of postsynaptic density proteins and its relation to synapse functions

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Postsynaptic density is an electron dense structure beneath the postsynaptic membrane of glutamatergic synapses. To reveal molecular mechanisms of its assembly, we performed the following experiments.1. We developed a novel method of estimating absolute number of PSD scaffolding molecules (PSD-95, GKAP, Shank, Homer) per single synapses. Based on the fluorescence intensity of microspheres calibarated against single GFP molecules, the number of GFP-tagged PSD molecules in single synapses was determined in cultured hippocampal neurons. Single synapses contain 100-400 of PSD molecules and the combined molecular mass of the four PSD molecules corresponds to 10% of the total mass of the PSD.2. RNA-binding protein TLS changes its distribution from dendritic shafts to spines along the course of dendritic maturation. TLS also translocates from the shaft cytoplasm to the spines by stimulation of metabotropic glutamate receptors. TLS-null neurons show abnormal development of spine morphology. These results suggest regulation of spine morphology by TLS after its activity-dependent transport into spines.3. We established multiple lines of transgenic mice expressing GFP-tagged PSD proteins. Using dissociated culture of hippocampal neurons derived from these transgenic mice, morphological changes of single spines were traced for more than a week, illustrating the transition of immature filopodia-like protrusions into mature spine-like structures. Long-term observation also revealed coordinated changes of PSD density in multiple dendrites of single neurons, suggesting integrative mechanism of synapse growth/elimination across different dendritic segments.In summary, these results indicate continual remodeling of scaffolding proteins, which serve as a major framework of the PSD structure and also suggest an important role of protein synthesis from dendritic mRNA in the maintenance of glutamatergic synapses.