Haubensak W, Narz F, Heumann R, Lessmann V
Lehrstuhl für Molekulare Neurobiochemie, Ruhr-Universität Bochum, NC7/170, Germany. volkmar.j.lessmann@ruhr
J Cell Sci. 1998 Jun;111 ( Pt 11):1483-93. doi: 10.1242/jcs.111.11.1483.
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.
哺乳动物神经营养因子蛋白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），可支持中枢神经系统和周围神经系统（CNS、PNS）中神经元的存活及表型。此外，最近发现外源性应用神经营养因子可调节啮齿动物中枢神经系统中的突触传递。然而，要证明神经营养因子作为突触传递的内源性快速作用调节剂的作用，需要展示神经营养因子的突触定位和分泌情况。我们现已构建了一种融合蛋白，其由N端的BDNF（啮齿动物海马体和新皮质中最丰富的神经营养因子）和C端的绿色荧光蛋白（GFP）组成，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，其在反式高尔基体网络（TGN）中的细胞定位、前体蛋白的加工以及成熟BDNF-GFP的分泌与未标记BDNF的特性并无差异。在原代大鼠皮质神经元瞬时转染后，发现BDNF-GFP存在于分泌调节途径的分泌颗粒中，这通过与分泌颗粒标记物嗜铬粒蛋白II的共定位得以表明。在转染神经元的神经突中发现了BDNF-GFP囊泡，其模式让人联想到未转染皮质神经元中内源性BDNF的定位。BDNF-GFP囊泡主要存在于神经元的树突体部分，而轴突定位则较少见。用突触素I抗体对突触终末进行免疫细胞化学染色显示，突触连接处附近BDNF-GFP囊泡的密度升高，表明BDNF的定位适合作为突触传递的急性调节剂发挥作用。这些数据表明，BDNF-GFP将是研究生理突触刺激期间BDNF突触释放的有用工具，从而使我们能够阐明神经营养因子释放参与依赖活动的突触可塑性的情况。
