Center of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, 3004-548 Coimbra, Portugal.
Neuroscience. 2010 Dec 29;171(4):981-92. doi: 10.1016/j.neuroscience.2010.10.019. Epub 2010 Oct 13.
A few studies have reported the existence of depletion of synaptic vesicles, and changes in neurotransmitter release and in the content of exocytotic proteins in the hippocampus of diabetic rats. Recently, we found that diabetes alters the levels of synaptic proteins in hippocampal nerve terminals. Hyperglycemia is considered the main trigger of diabetic complications, although other factors, such as low insulin levels, also contribute to diabetes-induced changes. Thus, the aim of this work was to evaluate whether long-term elevated glucose per se, which mimics prolonged hyperglycemia, induces significant changes in the content and localization of synaptic proteins involved in exocytosis in hippocampal neurons. Hippocampal cell cultures were cultured for 14 days and were exposed to high glucose (50 mM) or mannitol (osmotic control; 25 mM plus 25 mM glucose), for 7 days. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. The protein levels of vesicle-associated membrane protein-2 (VAMP-2), synaptosomal-associated protein-25 (SNAP-25), syntaxin-1, synapsin-1, synaptophysin, synaptotagmin-1, rabphilin 3a, and also of vesicular glutamate and GABA transporters (VGluT-1 and VGAT), were evaluated by immunoblotting, and its localization was analyzed by immunocytochemistry. The majority of the proteins were not affected. However, elevated glucose decreased the content of SNAP-25 and increased the content of synaptotagmin-1 and VGluT-1. Moreover, there was an accumulation of syntaxin-1, synaptotagmin-1 and VGluT-1 in the cell body of some hippocampal neurons exposed to high glucose. No changes were detected in mannitol-treated cells. In conclusion, elevated glucose per se did not induce significant changes in the content of the majority of the synaptic proteins studied in hippocampal cultures, with the exception of SNAP-25, synaptotagmin-1 and VGluT-1. However, there was an accumulation of some proteins in cell bodies of hippocampal neurons exposed to elevated glucose, suggesting that the trafficking of these proteins to the synapse may be compromised. Moreover, these results also suggest that other factors, in addition to hyperglycemia, certainly contribute to alterations detected in synaptic proteins in diabetic animals.
一些研究报告指出，糖尿病大鼠海马突触小泡耗竭，神经递质释放和外排蛋白含量发生变化。最近，我们发现糖尿病改变了海马神经末梢突触蛋白的水平。高血糖被认为是糖尿病并发症的主要触发因素，尽管其他因素，如胰岛素水平降低，也会导致糖尿病引起的变化。因此，本工作旨在评估长期高血糖本身（模拟长期高血糖）是否会导致海马神经元中参与胞吐作用的突触蛋白的含量和定位发生显著变化。海马细胞培养物培养 14 天，然后暴露于高葡萄糖（50mM）或甘露醇（渗透对照；25mM 加 25mM 葡萄糖）中 7 天。通过 MTT 和 Hoechst 测定分别评估细胞活力和核形态。通过免疫印迹评估囊泡相关膜蛋白-2（VAMP-2）、突触相关蛋白-25（SNAP-25）、突触融合蛋白-1、突触素-1、突触小泡谷氨酸和 GABA 转运体（VGluT-1 和 VGAT）的蛋白水平，并通过免疫细胞化学分析其定位。大多数蛋白质没有受到影响。然而，高葡萄糖降低了 SNAP-25 的含量，增加了突触小泡蛋白-1 和 VGluT-1 的含量。此外，暴露于高葡萄糖的一些海马神经元的胞体中积累了 syntaxin-1、synaptotagmin-1 和 VGluT-1。在甘露醇处理的细胞中未检测到变化。总之，高葡萄糖本身并未导致海马培养物中大多数研究的突触蛋白含量发生显著变化，除了 SNAP-25、synaptotagmin-1 和 VGluT-1。然而，暴露于高葡萄糖的海马神经元胞体中某些蛋白质的积累表明这些蛋白质向突触的转运可能受到损害。此外，这些结果还表明，除了高血糖之外，其他因素肯定会导致糖尿病动物突触蛋白的改变。
