Regulation of protein kinase C-mediated dopamine transporter endocytosis in vivo

描述(申请人提供)：中脑中的多巴胺能神经元控制情绪、认知和运动的各个方面，并参与包括药物成瘾在内的各种疾病的发病机制。多巴胺能神经传递的一个主要调节因子是多巴胺转运体，它负责将多巴胺重新摄取回突触前神经元。成瘾药物，如苯丙胺，不仅通过竞争性抑制转运，而且通过利用神经元的内源性信号和内吞途径下调细胞表面转运体的表达，破坏了多巴胺的重新摄取。先前的研究表明，多巴胺转运体表面表达的调控涉及许多信号通路和内吞通路。例如，已经观察到蛋白激酶C被其强大的激活剂佛波酯激活，从而增加了细胞系和啮齿动物神经元中转运体的内化。蛋白激酶鉴定转化过程中的关键障碍 作为进入治疗和/或诊断靶点的相关信号通路，缺乏对蛋白激酶C如何调节多巴胺神经元中的多巴胺转运体运输的机械性见解。虽然已知PKC刺激的DAT内吞作用依赖于DAT泛素化，但很少有机制细节被揭示，例如涉及PKC的异构体或内源性脑化合物刺激PKC介导的内吞作用。为了解决这一障碍，我将研究多巴胺转运体调节的机制，特别是在多巴胺神经元中，以阐明调节转运体内吞和泛素化的蛋白激酶C的异构体。初步研究表明，蛋白激酶C-epsilon在多巴胺神经元中的表达具有重要作用，这促使我提出了蛋白激酶C-epsilon调节多巴胺能神经元中多巴胺转运体内吞的假说。我将使用传统的神经化学和创新的方法来检验这一假设。例如，我将通过立体定向传递含有Lox-Stop-Lox转录停止盒的病毒载体，选择性地控制多巴胺转运体-Cre突变小鼠多巴胺能神经元中的基因功能。此外，我将通过分析在小鼠多巴胺神经元中外源表达的细胞外表位标记突变转运体的内吞作用来补充我们对内源性转运体运输的研究。我希望这些结果将为未来的机制和行为实验奠定基础，最终可能导致识别新的治疗靶点，以调节病理性多巴胺能信号。

DESCRIPTION (provided by applicant): Dopaminergic neurons in the midbrain control aspects of mood, cognition, and movement and are involved in the pathogenesis of a variety of disorders including drug addiction. A primary regulator of dopaminergic neurotransmission is the dopamine transporter, which is responsible for the re-uptake of dopamine back into the pre-synaptic neuron. Drugs of addiction, such as amphetamine, disrupt dopamine re-uptake not only via competitive inhibition of transport but also by harnessing the neuron's endogenous signaling and endocytic pathways to down-regulate transporter expression at the cell surface. Previous research has implicated a number of signaling and endocytic pathways in the regulation of dopamine transporter surface expression. For example, activation of protein kinase C by its potent activator phorbol ester has been observed to increase transporter internalization in cell lines and rodent neurons. The critical barrier in transforming the identification of protein kinase C as a relevant signaling pathway into a therapeutic and/or diagnostic target is the lack of mechanistic insight into how protein kinase C regulates dopamine transporter trafficking in dopamine neurons. While PKC-stimulated DAT endocytosis is known to be dependent on DAT ubiquitination, few mechanistic details have been revealed, such as the isoform of PKC involved or what endogenous brain compounds stimulate PKC-mediated endocytosis. To address this barrier to progress, I will examine mechanisms of dopamine transporter regulation specifically in dopamine neurons to clarify the isoform of protein kinase C that regulates transporter endocytosis and ubiquitination. Preliminary studies indicate an important role for protein kinase C-epsilon, an isoform expressed in dopamine neurons, prompting me to propose the hypothesis that protein kinase C-epsilon regulates dopamine transporter endocytosis in dopaminergic neurons. I will test this hypothesis by using traditional neurochemical as well as innovative approaches. For example, I will control gene function selectively in dopaminergic neurons of dopamine transporter-Cre mutant mice by stereotaxic delivery of viral vectors that contain a Lox-stop-Lox transcriptional stop cassette. Additionally, I will complement our studies of endogenous transporter trafficking by analyzing the endocytosis of an extracellular epitope-tagged mutant transporter exogenously expressed in mouse dopamine neurons. I expect that the results will lay the foundation for future mechanistic and behavioral experimentation that may eventually lead to the identification of new therapeutic targets for modulating pathological dopaminergic signaling.