Serotonin transporter-mediated regulation of neuroendocrine exocytosis

描述（申请人提供）：5 -羟色胺（5HT）是一种重要的神经调节剂，5 -羟色胺转运体（SERT）在控制5 -羟色胺信号传导和有效性中起着核心作用。SERT的遗传变异与几种神经精神疾病有关，包括抑郁症、焦虑症、强迫症和自闭症。此外，SERT是包括选择性血清素再摄取抑制剂（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尚不清楚。SERT也存在于外周，包括在肾上腺染色质细胞中显著表达，但其作用尚不清楚。染色质细胞是交感神经系统中重要的神经内分泌成分，它释放儿茶酚胺、多肽和其他递质的混合物，以确保协调的生理反应，例如在对急性压力的“战斗或逃跑”反应期间。它们还可以作为一个多功能的神经分泌模型，使详细了解胞吐的细胞和分子机制成为可能。虽然5HT通常被认为是通过细胞表面受体起作用，但最近有人提出它在平滑肌、血小板和胰腺β细胞中作为细胞内信号分子起作用。这种新机制涉及通过谷氨酰胺转酶-2（被称为“血清素化”）将5HT与胞质蛋白共价结合。我们的初步数据表明，染色质细胞通过SERT的摄取积累5HT。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在调节嗜铬细胞胞吐中的作用。我们假设5HT在SERT急性摄取后作为细胞内信使调节神经内分泌胞外分泌。我们将结合膜片钳、碳纤维电流计、钙成像和“关在”细胞内信使的光解与从野生型小鼠、SERT敲除小鼠和一种具有点突变的新型敲入小鼠分离的染色质细胞，该突变使SERT对抗抑郁药和可卡因不敏感。我们还将使用电子显微镜（EM）来评估大致密核囊泡的数量、大小和分布。生化方法将确定是否发生活性依赖的血清素化。这些研究有可能显著改变我们对控制神经分泌机制的理解，以及抗抑郁药和其他靶向SERT药物的机制。这将为未来扩展应用提供基础，以研究5HT / SERT介导的信号转导的新范例。

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.