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

体内蛋白激酶 C 介导的多巴胺转运蛋白内吞作用的调节

• 批准号: 8525510
• 负责人: ETHAN ROBERT BLOCK
• 金额: $ 5.39万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525510
• 关键词: Address; Amphetamines; Back; Behavioral; Binding; Biochemical; Brain; Cell Line; Cell membrane; Cell surface; Co-Immunoprecipitations; Cognition; Communication; Complement; Development; Diagnostic; Disease; Dominant-Negative Mutation; Dopamine; Drug Addiction; Endocytosis; Enzymes; Epitopes; Event; Foundations; Future; Goals; Knowledge; Lead; Learning; Mediating; Mental disorders; Midbrain structure; Moods; Movement; Mus; Mutant Strains Mice; Nerve; Neurons; Parkinson Disease; Pathogenesis; Pathway interactions; Phorbol Esters; Protein Isoforms; Protein Kinase C; Proteins; Purinoceptor; Regulation; Research; Research Proposals; Rodent; Role; Series; Signal Pathway; Signal Transduction; Surface; Synapses; Testing; Therapeutic; Ubiquitination; Viral Vector; Work; addiction; atypical protein kinase C; dopamine transporter; dopaminergic neuron; extracellular; gene function; in vivo; innovation; insight; mutant; neurochemistry; neurotransmission; new therapeutic target; novel; presynaptic; protein complex; protein kinase C epsilon; public health relevance; research study; therapeutic target; trafficking; uptake

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.

描述（由申请人提供）：中脑中的多巴胺能神经元控制情绪、认知和运动，并参与多种疾病（包括药物成瘾）的发病机制。多巴胺能神经传递的主要调节剂是多巴胺转运蛋白，其负责将多巴胺再摄取回到突触前神经元中。成瘾药物，如安非他明，破坏多巴胺的再摄取，不仅通过竞争性抑制运输，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的转运蛋白表达。先前的研究表明，多巴胺转运蛋白表面表达的调节涉及许多信号传导和内吞途径。例如，已经观察到蛋白激酶C通过其有效激活剂佛波醇酯的激活增加细胞系和啮齿动物神经元中的转运蛋白内化。蛋白激酶鉴定转变的关键障碍 蛋白激酶C作为相关信号通路进入治疗和/或诊断靶点的原因是缺乏对蛋白激酶C如何调节多巴胺神经元中多巴胺转运蛋白运输的机制性洞察。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，例如涉及的PKC亚型或内源性脑化合物刺激PKC介导的内吞作用。为了解决这一障碍的进展，我将研究多巴胺转运蛋白的调节机制，特别是在多巴胺神经元，以澄清蛋白激酶C的亚型，调节转运蛋白内吞和泛素化。初步研究表明，蛋白激酶C-β（一种在多巴胺神经元中表达的亚型）具有重要作用，这促使我提出蛋白激酶C-β调节多巴胺能神经元中多巴胺转运蛋白内吞作用的假设。我将通过使用传统的神经化学方法以及创新的方法来验证这一假设。例如，我将通过立体定向递送含有Lox-stop-Lox转录终止盒的病毒载体，在多巴胺转运蛋白Cre突变小鼠的多巴胺能神经元中选择性地控制基因功能。此外，我将通过分析小鼠多巴胺神经元中外源表达的细胞外表位标记的突变转运蛋白的内吞作用来补充我们对内源性转运蛋白转运的研究。我希望这些结果将为未来的机制和行为实验奠定基础，这些实验最终可能导致识别用于调节病理性多巴胺能信号传导的新治疗靶点。