Dynamics of postsynaptic density proteins and its relation to synapse functions

突触后致密物是突触后膜下的电子致密结构。为了揭示其组装的分子机制，我们进行了以下实验.我们开发了一种新的方法，估计PSD支架分子（PSD-95，GKAP，柄，荷马）的绝对数量每单个突触。基于针对单个GFP分子校准的微球的荧光强度，在培养的海马神经元中确定单个突触中GFP标记的PSD分子的数量。单个突触包含100-400个PSD分子，并且四个PSD分子的组合分子量对应于PSD总质量的10%。RNA结合蛋白TLS在树突成熟过程中从树突轴向树突棘的分布发生沿着变化。TLS还通过刺激代谢型谷氨酸受体从轴细胞质易位到棘。TLS-null神经元表现出异常的脊髓形态发育。这些结果表明TLS通过活性依赖性转运进入棘后对棘形态的调节.我们建立了表达GFP标记的PSD蛋白的转基因小鼠的多个品系。使用来自这些转基因小鼠的海马神经元的分离培养物，追踪单个棘的形态变化超过一周，说明未成熟的丝状伪足样突起向成熟的棘样结构的过渡。长期观察还发现单个神经元的多个树突中PSD密度的协调变化，提示突触生长/消除跨不同树突节段的整合机制。总之，这些结果表明，作为PSD结构的主要框架的支架蛋白的持续重构，也提示树突mRNA的蛋白合成在维持突触能突触中的重要作用。

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.