Department of Chemistry and Biological Science, School of Science and Engineering, Aoyama Gakuin University, Kanagawa, Japan.
J Neurosci Res. 2010 Oct;88(13):2911-22. doi: 10.1002/jnr.22457.
We have previously shown that repetitive exposures to glutamate (100 muM, 3 min, three times at 24-hr intervals) induced a long-lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (for repetitive LTP-induced synaptic enhancement). To investigate the molecular mechanisms underlying RISE, we first analyzed the time course of gene expression changes between 4 hr and 12 days after repetitive stimulation using an original oligonucleotide microarray: "synaptoarray." The results demonstrated that changes in the expression of synapse-related genes were induced in two time phases, an early phase of 24-96 hr and a late phase of 6-12 days after the third stimulation. Comprehensive screening at 48 hr after the third stimulation using commercially available high-density microarrays provided candidate genes responsible for RISE. From real-time PCR analysis of these and related genes, two categories of genes were identified, 1) genes previously reported to be induced by physiological as well as epileptic activity (bdnf, grm5, rgs2, syt4, ania4/carp/dclk) and 2) genes involved in cofilin-based regulation of actin filament dynamics (ywhaz, ssh1l, pak4, limk1, cfl). In the first category, synaptotagmin 4 showed a third stimulation-specific up-regulation also at the protein level. Five genes in the second category were coordinately up-regulated by the second stimulation, resulting in a decrease in cofilin phosphorylation and an enhancement of actin filament dynamics. In contrast, after the third stimulation, they were differentially regulated to increase cofilin phosphorylation and enhance actin polymerization, which may be a key step leading to the establishment of RISE.
我们之前已经表明，重复暴露于谷氨酸（100 μM，3 分钟，24 小时间隔重复三次）会诱导大鼠海马切片培养物中持久的突触增强，伴随着突触发生，这种现象称为 RISE（重复长时程增强诱导的突触增强）。为了研究 RISE 背后的分子机制，我们首先使用原始寡核苷酸微阵列“synaptoarray”分析了重复刺激后 4 小时至 12 天之间基因表达变化的时间过程。结果表明，突触相关基因的表达变化分为两个时相，即第三次刺激后 24-96 小时的早期和 6-12 天的晚期。第三次刺激后 48 小时使用商业上可用的高密度微阵列进行全面筛选，提供了负责 RISE 的候选基因。对这些和相关基因的实时 PCR 分析表明，有两类基因，1）先前报道的生理和癫痫活动诱导的基因（bdnf、grm5、rgs2、syt4、ania4/carp/dclk）和 2）参与肌动蛋白丝动力学的肌动蛋白丝动力学的基因（ywhaz、ssh1l、pak4、limk1、cfl）。在第一类中，突触结合蛋白 4 也在蛋白质水平上表现出第三次刺激特异性上调。第二类中的五个基因在第二次刺激下协同上调，导致肌动蛋白丝动力学的 cofilin 磷酸化减少和增强。相比之下，第三次刺激后，它们被差异调节以增加 cofilin 磷酸化并增强肌动蛋白聚合，这可能是建立 RISE 的关键步骤。
