Dopamine Transporter Endocytic Trafficking in Striatal Dopaminergic Neurons: Differential Dependence on Dynamin and the Actin Cytoskeleton
Dopamine Transporter Endocytic Trafficking in Striatal Dopaminergic Neurons: Differential Dependence on Dynamin and the Actin Cytoskeleton
复制标题
DOI：
10.1523/jneurosci.3284-13.2013
复制
发表时间：
2013-11-06
影响因子：
5.3
通讯作者：
Melikian, Haley E.
中科院分区：
文献类型：
--
作者：
Gabriel, Luke R.;Wu, Sijia;Melikian, Haley E.
多巴胺能信号深刻影响奖励行为、运动和执行功能。突触前多巴胺（DA）转运体（DAT）重新捕获释放的DA，从而限制突触DA的可用性并维持多巴胺能张力。DAT组成性内化，PKC激活迅速加速DAT内吞作用，导致DAT表面丢失。长期以来的证据支持在异源表达研究中pkc刺激的DAT运输。然而，在培养的多巴胺能神经元中，pkc刺激的DAT内化并不容易观察到。此外，相互矛盾的报告暗示经典和非经典内吞机制介导数据传输。先前的数据传输研究主要依赖于慢性基因破坏和显性阴性蛋白表达，或者在细胞系和培养的神经元中进行，其结果难以转化为成人多巴胺能神经元。在这里，我们使用新描述的动力蛋白抑制剂来测试成人多巴胺能神经元中构成型和pkc刺激的DAT内化是否依赖于动力蛋白。在小鼠纹状体切片中进行的离体生物素化研究表明，PKC的急性激活会导致天然DAT表面的丢失，并且表面DAT在愿意内吞和抵抗内吞的种群之间令人惊讶地划分。急性动力蛋白抑制表明，构成型DAT内化与动力蛋白无关，而pkc刺激的DAT内化则与动力蛋白相关。此外，全内反射荧光显微镜实验表明，本构性DAT内化等效地发生在脂筏和非筏微结构域，而pkc刺激的DAT内化仅发生在脂筏。最后，DAT内吞循环依赖于与肌动蛋白细胞骨架协同作用的动力依赖机制。这些研究首次全面研究了离体成年神经元的原生数据传输，并揭示了数据表面动力学是由复杂的多模式机制控制的。
Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.