Study on Parkinson's disease model rats with antisense-induced nigral knockdown of synaptotagmin I

突触结合蛋白I反义诱导黑质基因敲低的帕金森病模型大鼠研究

• 批准号: 13670665
• 负责人: SAJI Makoto
• 金额: $ 2.18万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670665/
• 关键词: antisense; synaptotagmin I; early Parkinson's disease; HVJ-liposome vector; dopamine depletion; nigral knockdown; motor dysfunction; abnormal synaptic plasticity; シナプトタグミン I; シナプトタグミン; パーキンソン病; 回転運動異常; 遺伝子発現抑制; ドーパミン放出; シンタキシン; 運動疾患

Purpose : We hypothesized that a pathogenesis for dyskinesias due to chronic L-DOPA treatment in patients with Parkinson's disease is not dopamine depletion itself but abnormal synaptic plasticity of striatal neurons induced by denervation of dopaminergic fibers. To test this hypothesis, in this study, we armed to produce a novel type of Parkinson's disease model animal that has dopamine depletion without denervation of dopaminergic fibers in the striatum by using antisense-induced nigral knockdown of synaptotagmin I, a regulatory protein of transmitter release machinery.Research results : 1) We examined by Western blot analysis whether the synaptotagmin I protein level in the hippocampus was down-regulated by antisense ODNs against synaptotagmin I (syt-AS) following the infra-hippocampal I injection of HVJ-liposome vector containing syt-AS. As a result, synaptotagmin level in the hippocampus was down-regulated maximally by 50 % at 4-8 days after the injection. And the down-regulation … More of synaptotagmin protein level by antisense-treatment was sustained for about 10 days. 2) Next, using the HVJ-liposome vector containing this effective antisense ODNs, we produced rats with nigral knockdown of synaptotagmin I by the infra-nigral injection of HVJ-liposome containing syt-AS. Four days after the infra-nigral injection of syt AS, rats with antisense-induced nigral knockdown of synaptotagmin I displayed slight motor dysfunction, rotation responses, in the amphetamine-Induced hyperlocomotion. 3) One day after the behavioral test for motor dysfunction, the rats with unilateral nigral knockdown of synaptotagmin I received bilaterally the implantation of micro-dialysis probe in the striatum. By measurement of dopamine level through the micro-dialysis probes, marked decrease (20-70%) of the amphetamine-induced release of dopamine was observed in the treated side of the striatum, compared with that in the non-treated side. 4) Unlike the 6-OHDA nigral lesion, the antisense-induced nigral knockdown did not cause apomorphine-induecd high expression of c-fos gene, an prominent indicator of abnormal synaptic plasticity, in the dopamine-depleted striatal neurons.Conclusion : As expected, antisense-induced nigral knockdown of synaptotagmin I produced marked reduction of dopamine release in the striatum. This reduction of dopamine release in the striatum was related to slight motor dysfunction, but the relationship between the dopamine depletion and the motor dysfunction was not linear. From these results, we conclude that rat with synaptotagmin nigral knockdown may be a model animal for early Parkinson's disease. Less

目的：我们假设帕金森病患者长期左旋多巴治疗引起的运动障碍的发病机制不是多巴胺耗竭本身，而是多巴胺能纤维去神经支配诱导的纹状体神经元突触可塑性异常。为了验证这一假设，在本研究中，我们使用反义诱导的黑质敲除突触结合蛋白I（一种递质释放机制的调节蛋白），制备了一种新型的帕金森病模型动物，该模型动物具有多巴胺耗竭，但纹状体中的多巴胺能纤维不受神经支配。研究结果：1）通过Western blot分析，我们检测了海马中突触结合蛋白I的蛋白水平是否被针对突触结合蛋白I的反义ODNs（syt-AS）下调。海马I注射含有syt-AS的HVJ-脂质体载体。结果，海马中的突触结合蛋白水平在注射后4-8天最大下调50%。而下调 ...更多信息 反义处理后突触结合蛋白水平的下降持续约10天。2)接着，使用含有这种有效的反义ODNs的HVJ-脂质体载体，我们通过黑质下注射含有syt-AS的HVJ-脂质体来产生黑质敲低突触结合蛋白I的大鼠。黑质下注射syt AS后4天，反义诱导的黑质突触结合蛋白I敲低的大鼠在苯丙胺诱导的过度运动中表现出轻微的运动功能障碍，旋转反应。3)在运动功能障碍的行为学测试后1天，单侧黑质突触结合蛋白I敲低的大鼠接受双侧纹状体微透析探针植入。通过微透析探针测量多巴胺水平，观察到与未处理侧相比，在处理侧纹状体中安非他明诱导的多巴胺释放显著减少（20-70%）。4)与6-OHDA黑质损伤不同，反义诱导的黑质敲除并没有引起阿朴吗啡诱导的c-fos基因的高表达，这是突触可塑性异常的一个突出指标，在多巴胺耗尽的纹状体neurons.Conclusion：正如预期的那样，反义诱导的黑质敲除synaptotagmin I引起纹状体多巴胺释放的显着减少。纹状体多巴胺释放的减少与轻微的运动功能障碍有关，但多巴胺耗竭与运动功能障碍之间的关系不是线性的。从这些结果，我们得出结论，突触结合蛋白黑质敲低大鼠可能是早期帕金森病的模型动物。少

项目成果

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