Dynamics of postsynaptic density proteins and its relation to synapse functions

突触后密度是谷氨酸突触突触后膜下的电子密度结构。为了揭示其组装的分子机制，我们进行了以下实验。我们开发了一种估算单个突触中PSD支架分子（PSD-95， GKAP, Shank, Homer）绝对数量的新方法。根据单个GFP分子标定的微球荧光强度，测定培养海马神经元中单个突触中GFP标记的PSD分子的数量。单个突触含有100-400个PSD分子，4个PSD分子的总分子质量相当于PSD总质量的10%。rna结合蛋白TLS在树突成熟过程中由树突轴向树突棘的分布发生变化。TLS也通过刺激代谢性谷氨酸受体从轴细胞质转移到棘。TLS-null神经元表现为脊柱形态发育异常。这些结果表明，TLS通过活动依赖性转运进入脊柱后，对脊柱形态有调控作用。我们建立了多系表达gfp标记的PSD蛋白的转基因小鼠。利用这些转基因小鼠的海马神经元分离培养，对单个棘的形态变化进行了一周多的追踪，说明了未成熟的丝状足样突起向成熟的棘样结构的转变。长期观察还发现单个神经元多树突内PSD密度的协同变化，提示不同树突节段间突触生长/消除的一体化机制。综上所述，这些结果表明，作为PSD结构主要框架的支架蛋白的持续重塑，也表明树突mRNA的蛋白质合成在维持谷氨酸突触中起着重要作用。

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.