Serotonin transporter-mediated regulation of neuroendocrine exocytosis

描述(申请人提供)：5-羟色胺(5-羟色胺)是一种重要的神经调节剂，5-羟色胺转运体(SERT)在控制5-羟色胺信号和可用性方面起着核心作用。SERT的基因变异与几种神经精神疾病有关，包括抑郁、焦虑、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)在内的抗抑郁药物的临床相关靶点，尽管这些药物的确切治疗机制尚不清楚。SERT也在外周发现，包括在肾上腺嗜铬细胞中的显著表达，其作用尚不清楚。嗜铬细胞是交感神经系统中一个重要的神经内分泌成分，它释放儿茶酚胺、多肽和其他递质的鸡尾酒，以确保协调的生理反应，例如在对急性应激的“战斗或逃跑”反应中。它们也是一个多功能的神经分泌模型，能够详细地了解胞吐作用的细胞和分子机制。虽然5-羟色胺通常被认为是通过细胞表面受体起作用的，但最近有人提出它在平滑肌、血小板和胰岛β细胞中作为细胞内信号分子发挥作用。这一新的机制包括通过转谷氨酰胺酶-2将5-羟色胺与胞浆蛋白共价结合(称为“5-羟色胺基化”)。我们的初步数据显示，嗜铬细胞通过SERT摄取5-羟色胺。我们使用药理学(SSRI)和转基因小鼠(SERT的敲除和敲入模型)的数据也支持SERT在调节嗜铬细胞胞吐中的作用。我们推测5-羟色胺在SERT急性摄取后作为细胞内信使调节神经内分泌的胞吐作用。我们将结合膜片钳、碳纤维安培、钙成像和细胞内信使的光解与从野生型小鼠、SERT基因敲除小鼠和一种新的点突变使SERT对抗抑郁药物和可卡因不敏感的敲入小鼠中分离出的嗜铬细胞。我们还将使用电子显微镜(EM)来评估大而致密的核心小泡的数量、大小和分布。生化方法将确定是否发生活性依赖的5-羟色胺基化。这些研究有可能极大地改变我们对控制神经分泌的机制以及抗抑郁药物和其他针对SERT的药物的机制的理解。这将为进一步研究5HT/SERT介导的信号转导的新范式奠定基础。

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.