Serotonin transporter-mediated regulation of neuroendocrine exocytosis

描述（由申请人提供）：5-羟色胺（5-HT）是一种重要的神经调质，5-羟色胺转运蛋白（SERT）在控制5-HT信号传导和可用性方面发挥核心作用。SERT的遗传变异与几种神经精神障碍有关，包括抑郁症、焦虑症、强迫症和自闭症。此外，SERT是包括选择性5-羟色胺再摄取抑制剂（SSRI）在内的抗抑郁药的临床相关靶点，尽管这些药物的确切治疗机制仍不清楚。SERT也存在于外周，包括肾上腺嗜铬细胞中的显著表达，其中其作用知之甚少。嗜铬细胞是交感神经系统的重要神经内分泌成分，其释放儿茶酚胺、肽和其他递质的混合物以确保协调的生理反应，例如在对急性应激的“战斗或逃跑”反应期间。它们也作为一个通用的神经分泌模型，使详细了解胞吐作用的细胞和分子机制。虽然5 HT通常被认为通过细胞表面受体起作用，但最近已提出其在平滑肌、血小板和胰腺β细胞中充当细胞内信号传导分子。这种新的机制涉及5 HT通过转氨酶-2（被称为“羟丙酰化”）与胞质蛋白的共价结合。我们的初步数据表明，嗜铬细胞积累5 HT通过摄取SERT。我们使用药理学（SSRIs）和转基因小鼠（SERT敲除和敲入模型）的数据也支持SERT在嗜铬细胞胞吐调节中的作用。我们推测，5-HT作为细胞内信使急性摄取SERT调节神经内分泌胞吐。我们将联合收割机膜片钳，碳纤维安培法，钙成像，和“笼”的细胞内信使与嗜铬细胞分离的野生型小鼠，SERT敲除小鼠，和一个新的敲入小鼠的点突变，使SERT不敏感的抗抑郁药和可卡因的光解。我们还将使用电子显微镜（EM）来评估大的致密核心囊泡的数量，大小和分布。生物化学方法将确定是否发生活性依赖性腺苷酰化。这些研究有可能显著改变我们对控制神经分泌的机制以及抗抑郁药和其他靶向SERT的药物的机制的理解。这将为未来扩展应用提供基础，以研究5 HT/ SERT介导的信号转导的新范式。

DESCRIPTION (provided by applicant): Serotonin (5HT) is an important neuromodulator, and the serotonin transporter (SERT) plays a central role in controlling 5HT signaling and availability. Genetic variations in SERT are associated with several neuropsychiatric disorders including depression, anxiety, obsessive compulsive disorder, and autism. Moreover, SERT is a clinically relevant target for antidepressants including selective serotonin reuptake inhibitors (SSRI's), although the precise therapeutic mechanisms of these drugs remain unclear. SERT is also found in the periphery, including prominent expression in adrenal chromaffin cells where its role is poorly understood. Chromaffin cells are an important neuroendocrine component of the sympathetic nervous system that release a cocktail of catecholamines, peptides, and others transmitters to ensure coordinated physiological responses, for example during the "fight-or-flight" response to acute stress. They also serve as a versatile neurosecretory model that enables detailed insight into the cellular and molecular mechanisms of exocytosis. Although 5HT is typically thought to act through cell surface receptors, recently it has been proposed to act as an intracellular signaling molecule in smooth muscle, platelets, and pancreatic beta-cells. This novel mechanism involves covalent binding of 5HT to cytosolic proteins by transglutaminase-2 (dubbed "serotonylation"). Our preliminary data show that chromaffin cells accumulate 5HT through uptake by SERT. Our data using pharmacology (SSRIs) and transgenic mice (knock-out and knock-in models of SERT) also support a role for SERT in the regulation of chromaffin cell exocytosis. We postulate that 5HT acts at as an intracellular messenger following acute uptake by SERT to modulate neuroendocrine exocytosis. We will combine patch-clamp, carbon fiber amperometry, calcium imaging, and photolysis of "caged" intracellular messengers with chromaffin cells isolated from wild type mice, SERT knockout mice, and a novel knock-in mouse with a point mutation that renders SERT insensitive to antidepressants and cocaine. We will also use electron microscopy (EM) to assess the number, size, and distribution of large dense core vesicles. Biochemical approaches will determine if activity dependent serotonylation occurs. These investigations have the potential to significantly change our understanding of the mechanisms that control neurosecretion and the mechanisms of antidepressants and other drugs that target SERT. This will provide the foundation for future expanded applications to investigate a new paradigm for 5HT / SERT mediated signal transduction.