Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments
Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments
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DOI：
10.1523/jneurosci.0744-20.2020
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发表时间：
2021-01-13
影响因子：
5.3
通讯作者：
Sorkin, Alexander
中科院分区：
文献类型：
--
作者：
Bagalkot, Tarique R.;Block, Ethan R.;Sorkin, Alexander
多巴胺转运蛋白（DAT）通过调节突触释放的多巴胺来控制多巴胺的神经传递。然而，运输路线的DAT，决定其突触附近的细胞表面浓度，是很差的特点。特别是不知道DAT是如何在空间上遥远的中脑体树突和纹状体轴突室之间运输的。为了检查这种“长距离”运输，在完整脑和急性脑切片中使用共聚焦、超分辨率和EM分析了HA表位标记的DAT在敲入小鼠（两种性别）的内侧前脑束（MFB）中的定位和膜扩散。HA-DAT是丰富的MFB轴突的质膜，类似于纹状体，虽然在MFB的HA-DAT的细胞内部分是更实质性的。细胞内HA-DAT与VPS 35共定位在轴突子集中，VPS 35是逆转录聚合物复合物的一个亚基，介导内体的回收。晚期内体、溶酶体和内质网在索马中丰富，但在MFB轴突中存在最少，表明DAT的生物合成和溶酶体降解仅限于索马。总之，这些数据表明，膜扩散是通过MFB的远程DAT运输的主要模式，虽然泡状交通的贡献可以是显着的MFB轴突的人口。基于HA-DAT扩散速率，MFB轴突中的质膜DAT以类似于20 d的半衰期翻转，这解释了DAT蛋白在脑中的极慢的翻转。出乎意料的是，DAT标记的MFB轴突的平均直径被观察到比纹状体报道的大两倍。这一发现的影响多巴胺神经元生理进行了讨论。
Dopamine transporter (DAT) controls dopamine neurotransmission by dearing synaptically released dopamine. However, traffidcing itineraries of DAT, which determine its cell-surface concentration near synapses, are poorly characterized. It is especially unknown how DAT is transported between spatially distant midbrain somatodendritic and striatal axonal compartments. To examine this "long-range" trafficking, the localization and membrane diffusion of HA-epitope tagged DAT in the medial forebrain bundle (MFB) of a knock-in mouse (both sexes) were analyzed using confocal, super-resolution and EM in intact brain and acute brain slices. HA-DAT was abundant in the plasma membrane of MFB axons, similar to the striatum, although the intracellular fraction of HA-DAT in MFB was more substantial. Intracellular HA-DAT colocalized with VPS35, a subunit of the retromer complex mediating recycling from endosomes, in a subset of axons. Late endosomes, lysosomes, and endoplasmic reticulum were abundant in the soma but minimally present in MFB axons, suggesting that biosynthesis and lysosomal degradation of DAT are confined to soma. Together, the data suggest that membrane diffusion is the main mode of long-range DAT transport through MFB, although the contribution of vesicular traffic can be significant in a population of MFB axons. Based on HA-DAT diffusion rates, plasma membrane DAT in MFB axons turns over with a halftime of similar to 20d, which explains the extremely slow turnover of DAT protein in the brain. Unexpectedly, the mean diameter of DAT-labeled MFB axons was observed to be twice larger than reported for striatum. The implications of this finding for dopamine neuron physiology are discussed.