Pantazis N J, Zaheer A, Dai D, Zaheer S, Green S H, Lim R
Department of Anatomy and Cell Biology, University of Iowa, College of Medicine, Bowen Science Building, Iowa City, IA 52242, USA.
Brain Res. 2000 May 19;865(1):59-76. doi: 10.1016/s0006-8993(00)02194-6.
Glial cells play active roles in neuronal survival, as well as neuroprotection against toxic insult. Recent studies suggest that the brain protein glia maturation factor (GMF) is involved in intracellular signaling in glia. This study investigated whether or not GMF plays a role in the survival-promoting and neuroprotective functions of glia. C6 glioma cells were transfected in vitro with GMF utilizing an adenovirus vector. The transfected cells overexpressed GMF intracellularly, but did not secrete the protein. The conditioned medium (CM) was obtained from the GMF-transfected cells (CM-GMF) and tested on primary neuronal cultures, consisting of cerebellar granule cells (CGC). The CGC cultures were utilized because these cultures have a background level of cell death, and the survival-promoting, i.e. neurotrophic effect, of the CM could be tested. In addition, since CGC cultures are ethanol-sensitive (ethanol enhances neuronal death), the neuroprotective effect of the CM against ethanol-induced cell death was tested also. We demonstrated that the CM-GMF had an enhanced neurotrophic effect as well as an increased neuroprotective effect against ethanol-induced cell death compared to control CM obtained from untransfected C6 cells (CM-Mock) or CM obtained from cells transfected with an unrelated gene (CM-LacZ). Because neurotrophins have trophic and protective effects, we investigated whether GMF-transfection upregulated the expression of neurotrophins in C6 cells. RT-PCR verified that GMF-transfected C6 cells had increased mRNA levels for BDNF and NGF. Immunoblotting corroborated the RT-PCR results and indicated that CM-GMF contained greater concentrations of BDNF and NGF protein compared to CM-Mock and CM-LacZ. A soluble TrkB-IgG fusion protein, which selectively binds BDNF and prevents its binding to the neuronal TrkB receptor, eliminated the neurotrophic effect of CM-GMF; whereas anti-NGF antibody was ineffective in preventing this effect, suggesting that the neurotrophic effect was due to BDNF. On the other hand, both the TrkB-IgG fusion protein and anti-NGF reduced neuroprotection, suggesting that BDNF and NGF both contribute to the neuroprotective effect of CM-GMF. In conclusion, GMF upregulates the expression of BDNF and NGF in C6 cells, and these factors exert neurotrophic and neuroprotective functions on primary neurons.
神经胶质细胞在神经元存活以及抵御毒性损伤的神经保护过程中发挥着积极作用。近期研究表明，脑蛋白胶质细胞成熟因子（GMF）参与神经胶质细胞的细胞内信号传导。本研究调查了GMF是否在神经胶质细胞的促存活和神经保护功能中发挥作用。利用腺病毒载体在体外将GMF转染至C6胶质瘤细胞。转染后的细胞在细胞内过表达GMF，但不分泌该蛋白。从转染GMF的细胞（CM-GMF）中获取条件培养基（CM），并在由小脑颗粒细胞（CGC）组成的原代神经元培养物上进行测试。之所以使用CGC培养物，是因为这些培养物存在一定程度的细胞死亡背景，从而可以测试CM的促存活作用，即神经营养作用。此外，由于CGC培养物对乙醇敏感（乙醇会加剧神经元死亡），因此也测试了CM对乙醇诱导的细胞死亡的神经保护作用。我们证明，与从未转染的C6细胞获得的对照CM（CM-Mock）或从转染无关基因的细胞获得的CM（CM-LacZ）相比，CM-GMF具有更强的神经营养作用以及对乙醇诱导的细胞死亡更强的神经保护作用。由于神经营养因子具有营养和保护作用，我们研究了GMF转染是否上调了C6细胞中神经营养因子的表达。逆转录聚合酶链反应（RT-PCR）证实，转染GMF的C6细胞中脑源性神经营养因子（BDNF）和神经生长因子（NGF）的mRNA水平升高。免疫印迹法证实了RT-PCR结果，并表明与CM-Mock和CM-LacZ相比，CM-GMF含有更高浓度的BDNF和NGF蛋白。一种可溶性的TrkB-IgG融合蛋白，它能选择性结合BDNF并阻止其与神经元TrkB受体结合，消除了CM-GMF的神经营养作用；而抗NGF抗体在阻止这种作用方面无效，这表明神经营养作用是由BDNF引起的。另一方面，TrkB-IgG融合蛋白和抗NGF都降低了神经保护作用，这表明BDNF和NGF都对CM-GMF的神经保护作用有贡献。总之，GMF上调了C6细胞中BDNF和NGF的表达，并且这些因子对原代神经元发挥神经营养和神经保护功能。
