Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh 15261, Pennsylvania.
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh 15261, Pennsylvania
eNeuro. 2024 Jan 29;11(1). doi: 10.1523/ENEURO.0491-23.2023. Print 2024 Jan.
Dopamine transporter (DAT) controls dopamine signaling in the brain through the reuptake of synaptically released dopamine. DAT is a target of abused psychostimulants such as amphetamine (Amph). Acute Amph administration induces transient DAT endocytosis, which, among other Amph effects on dopaminergic neurons, elevates extracellular dopamine. However, the effects of repeated Amph abuse, leading to behavioral sensitization and drug addiction, on DAT are unknown. Hence, we developed a 14 d Amph-sensitization protocol in knock-in mice expressing HA-epitope-tagged DAT (HA-DAT) and investigated the effects of Amph challenge on sensitized HA-DAT animals. The Amph challenge resulted in the highest locomotor activity on Day 14 in both sexes, which was sustained for 1 h in male but not female mice. Strikingly, significant (by 30-60%) loss of the HA-DAT protein in the striatum was caused by the Amph challenge of sensitized males but not females. Amph also reduced of dopamine transport in the striatal synaptosomes of males without changing values. Consistently, immunofluorescence microscopy revealed a significant increase of HA-DAT colocalization with the endosomal protein VPS35 only in Amph-challenged males. Amph-induced loss of striatal HA-DAT in sensitized mice was blocked by chloroquine, vacuolin-1, and inhibitor of Rho-associated kinases ROCK1/2, indicative of the involvement of endocytic trafficking in the DAT protein loss. Interestingly, an apparent degradation of HA-DAT protein was observed in the nucleus accumbens and not in the dorsal striatum. We propose that Amph challenge in sensitized mice triggers Rho-mediated endocytosis and post-endocytic trafficking of DAT in a brain-region-specific and sex-dependent manner.
多巴胺转运体（DAT）通过突触间隙多巴胺的再摄取来控制大脑中的多巴胺信号。DAT 是滥用精神兴奋剂（如安非他命）的靶点。急性安非他命给药会诱导 DAT 的瞬时内吞作用，这除了对多巴胺能神经元的其他安非他命作用外，还会升高细胞外多巴胺。然而，反复滥用安非他命导致行为敏化和成瘾对 DAT 的影响尚不清楚。因此，我们在表达 HA 表位标签的 DAT（HA-DAT）的基因敲入小鼠中开发了一个 14 天的安非他命敏化方案，并研究了安非他命挑战对敏化的 HA-DAT 动物的影响。安非他命挑战在两性中均导致最高的运动活性，在雄性中持续 1 小时，但在雌性中不持续。引人注目的是，安非他命挑战敏化雄性但不雌性小鼠导致纹状体中的 HA-DAT 蛋白显著（30-60%）丢失。安非他命还降低了雄性纹状体突触小体中多巴胺转运的 ，但不改变 值。一致地，免疫荧光显微镜显示，仅在安非他命挑战的雄性中，HA-DAT 与内体蛋白 VPS35 的共定位显著增加。氯喹、 vacuolin-1 和 Rho 相关激酶 ROCK1/2 的抑制剂阻断了敏化小鼠中纹状体 HA-DAT 的安非他命诱导丢失，表明内吞作用参与了 DAT 蛋白的丢失。有趣的是，在伏隔核中观察到明显的 HA-DAT 蛋白降解，而在背侧纹状体中没有。我们提出，敏化小鼠中的安非他命挑战以脑区特异性和性别依赖性的方式触发 Rho 介导的内吞作用和 DAT 的内吞后转运。
