Aberrant trafficking of the high‐affinity choline transporter in AP‐3‐deficient mice
Aberrant trafficking of the high‐affinity choline transporter in AP‐3‐deficient mice
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DOI：
10.1111/j.1460-9568.2008.06268.x
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发表时间：
2008-06
影响因子：
3.4
通讯作者：
H. Misawa;Hirofumi Fujigaya;Takashi Nishimura;Y. Moriwaki;T. Okuda;K. Kawashima;K. Nakata;A. Ruggiero-A.-R
中科院分区：
文献类型：
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作者：
H. Misawa;Hirofumi Fujigaya;Takashi Nishimura;Y. Moriwaki;T. Okuda;K. Kawashima;K. Nakata;A. Ruggiero-A.-R
高亲和力胆碱转运蛋白（CHT）在胆碱能神经元中表达，并有效地转运到轴突终末，在那里它控制乙酰胆碱合成的限速步骤。最近的研究表明，大多数CHT出乎意料地位于突触囊泡（SV）而不是突触前质膜上，建立了囊泡CHT运输作为活性依赖性CHT调节的基础。在这里，我们分析了CHT在衔接蛋白-3（AP-3）缺陷小鼠模型摩卡中的细胞内分布。在摩卡小鼠中，细胞体中的颗粒状结构被CHT抗体强烈标记，表明CHT从细胞体到轴突末端的运输可能存在缺陷。蛋白质印迹分析显示，与野生型小鼠相比，摩卡小鼠SV上的CHT降低了30%。然而，没有显着差异，突触体胆碱摄取活性检测，与CHT的存在一个大的水库池。为了进一步表征CHT运输，我们建立了PC 12 D-CHT细胞系。在这条线中，发现CHT与突触素阳性突触样微泡（SLMV）亚群相关。通过用阻止AP依赖性膜运输的试剂处理细胞，SLMV上检测到的CHT的量大大减少。这些结果表明，AP对CHT在神经细胞中的运输具有重要功能。
The high‐affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate‐limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity‐dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein‐3 (AP‐3)‐deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild‐type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D‐CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin‐positive synaptic‐like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP‐dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.