MDM2在啮齿动物脑神经元轴突起始段的分布和表达模式

Localization and expression pattern of MDM2 in axon initial segments of neuron in rodent brain

本文旨在研究鼠双微体基因2(murine double minute 2,MDM2)在啮齿动物脑内的分布及表达模式。应用免疫组化、免疫荧光双标及免疫印迹等技术检测MDM2蛋白在出生后不同阶段昆明小鼠大脑皮层和海马内的表达,及其在成年SD大鼠脑内和原代培养神经元中的分布,观察MDM2与神经元轴突起始段(axon initial segment,AIS)标志蛋白在分布上的相关性。进而采用MDM2抑制剂Nutlin-3在SD大鼠海马内注射,检测MDM2的分布及Nav1.6分布变化。结果显示,昆明小鼠出生后不同时程MDM2在皮层和海马的表达呈现不同的变化模式,但都显示随发育的进展,MDM2逐渐向AIS富集,这一现象在出生7天后逐渐显著。在成年SD大鼠不同脑区的神经元和原代培养神经元中也观察到MDM2富集于AIS的现象,并且其与AIS标志蛋白AnkG、Nav1.6共定位。脑内注射Nutlin-3不仅抑制MDM2在海马神经元AIS的分布,还导致Nav1.6在AIS的分布减少,并且树突标志物MAP2在神经元中的极性分布也发生改变。结果提示MDM2蛋白可在正常成年啮齿类动物脑神经元AIS富集表达,对维持AIS特性和神经元极性起调节作用。

To investigate the murine double minute 2 （MDM2） localization and expression pattern in brain, immunohistochemistry, immunofluorescent staining and immunoblotting methods were used to analyze it in brains of Kunming mice during postnatal devel- opment, in brains of adult SD rats and in primarily cultured neurons. The distribution of MDM2 and markers of axon initial segment （AIS） was analyzed by double immunolabeling. In addition, Nutlin-3, a MDM2 antagonist, was injected into hippocampus to analyze the effect on the distribution of MDM2 and AIS protein Navl.6 in AIS. The results showed that the dynamic expression patterns of MDM2 protein in cerebral cortex and hippocampus of Kunming mice after birth were different. However, it was similar that MDM2 was gradually enriched to AIS during postnatal development, especially after postnatal day 7. The MDM2 in AIS was also observed in different brain regions of adult SD rat brain and in primarily cultured neurons, where MDM2 was colocalized with AIS markers such as AnkG and Nav 1.6. In addition, hippocampal injection of Nutlin-3 could induce the loss of the characteristic distribution of MDM2 in AIS. Moreover, Nutlin-3 not only caused a decrease of Navl.6 distributing in AIS, but also disrupted the polarized distribution of MAP2 in neurons. These results indicate that MDM2 can be enriched at the AIS of adult rodent brain, which might play a role in regu- lation of the maintenance ofAIS fimction and neuronal polarity.