BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons.
BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons.
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发表时间：
1998-06
影响因子：
4
通讯作者：
W. Haubensak;F. Narz;R. Heumann;V. Lessmann
中科院分区：
文献类型：
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作者：
W. Haubensak;F. Narz;R. Heumann;V. Lessmann
哺乳动物神经营养因子的蛋白质家族包括神经生长因子(NGF)、脑源性神经营养因子(BDNF)、神经营养因子-3和-4/5(NT-3、NT-4/5)，支持中枢和外周神经系统(CNS、PNS)神经元的存活和表型。此外，最近发现外源性应用神经营养素可以调节啮齿动物中枢神经系统中的突触传递。然而，为了证明神经拓扑素作为突触传递的内源性快速作用调节器的作用，突触的定位和神经营养因子的分泌需要被证明。我们现在已经构建了由N端BDNF(啮齿动物海马和新皮质中含量最丰富的神经营养因子)和C端绿色荧光蛋白(GFP)组成的融合蛋白，以阐明BDNF在皮质神经元中的细胞定位。BDNF-GFP在COS-7细胞中的瞬时表达表明，细胞在反式高尔基体网络(TGN)中的定位、前体蛋白的加工和成熟BDNF-GFP的分泌与未标记的BDNF的性质没有区别。瞬时转染大鼠皮质神经元后，BDNF-GFP出现在分泌调节途径的分泌颗粒中，与分泌颗粒标记物Secure-Granin II共定位。在转基因神经元的轴突中发现BDNF-GFP囊泡，其模式与内源性BDNF在未转染的皮质神经元中的定位相似。BDNF-GFP囊泡主要分布在神经元的体树突间，但较少发现附加轴突定位。突触蛋白I抗体免疫细胞化学染色显示，突触连接附近的BDNF-GFP囊泡密度增加，表明BDNF作为突触传递的急性调节器具有适当的定位。这些数据表明，BDNF-GFP将是研究生理突触刺激过程中突触释放BDNF的有用工具，从而使我们能够阐明神经营养因子释放在活性依赖性突触可塑性中的参与。
The protein family of mammalian neurotrophins, comprising nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and -4/5 (NT-3, NT-4/5), supports the survival and the phenotype of neurons from the central as well as the peripheral nervous system (CNS, PNS). In addition, exogenous application of neurotrophins has recently been found to modulate synaptic transmission in the rodent CNS. However, to provide evidence for a role of neurotophins as endogenous fast acting modulators of synaptic transmission, the synaptic localization and secretion of neurotrophins needs to be shown. We have now constructed a fusion protein consisting of N-terminal BDNF (the most abundant neurotrophin in the rodent hippocampus and neocortex) and C-terminal green fluorescent protein (GFP) to elucidate the cellular localization of BDNF in cortical neurons. Transient expression of BDNF-GFP in COS-7 cells revealed that the cellular localization in the trans-Golgi network (TGN), the processing of precursor proteins and the secretion of mature BDNF-GFP is indistinguishable from the properties of untagged BDNF. Upon transient transfection of primary rat cortical neurons, BDNF-GFP was found in secretory granules of the regulated pathway of secretion, as indicated by colocalization with the secretory granule marker secretogranin II. BDNF-GFP vesicles were found in the neurites of transfected neurons with a pattern reminiscent of the localization of endogenous BDNF in untransfected cortical neurons. BDNF-GFP vesicles were found predominantly in the somatodendritic compartment of the neurons, whereas additional axonal localization was found less frequently. Immunocytochemical staining of synaptic terminals with synapsin I antibodies revealed that the density of BDNF-GFP vesicles is elevated in the vicinity of synaptic junctions, indicating that BDNF is localized appropriately to function as an acute modulator of synaptic transmission. These data suggest that BDNF-GFP will be a useful tool to investigate synaptic release of BDNF during physiological synaptic stimulation, and will thereby allow us to elucidate the participation of neurotrophin release in activity dependent synaptic plasticity.