The Norepinephrine Transporter as a Novel Target for a Bone Anabolic Treatment

描述（由申请人提供）：本申请中提出的研究的长期目标是设计一种新的骨合成代谢治疗方法。抗抑郁药，根据其选择性，针对神经元中的血清素转运体（SERT）或去甲肾上腺素转运体（NET）。血清素转运蛋白家族抗抑郁药物的使用与骨质流失和骨折风险增加有关。相比之下，NET家族的抗抑郁药，尽管具有较弱的血清素转运体选择性，但在一项临床研究中并未显示出对骨骼的负面影响，甚至似乎可以降低骨折风险。我们的初步数据表明，小鼠体内NET的遗传阻断会引起骨体积的急剧增加，这是由骨吸收减少和骨形成增加引起的。Net在神经细胞中表达，其功能是从突触中清除去甲肾上腺素（NE），但令人惊讶的是，它也在成骨细胞中表达，其功能尚未被探索。我们在之前的研究中已经证明，交感神经通过β 2肾上腺素能受体（¿2AR）释放的NE会导致骨质流失。基于这些研究，我们可以预期，缺乏NET的小鼠的特征是突触中NE的积累，从而导致骨质流失。我们在Net缺陷小鼠中发现的相反表型使我们提出了三种可能的Net缺陷导致高骨量的机制：a)成骨细胞的慢性¿2AR刺激导致受体脱敏和交感神经抗成骨信号的阻断，b)大脑中抑制性a2AR刺激引起的交感神经张力降低，和/或c) Net对成骨细胞分化或功能的细胞自主抑制功能。无论其作用机制如何，我们的初步结果确定NET是一种新型骨合成代谢药物发现的潜在分子靶点，并使我们假设临床可用的选择性NET阻滞剂可以促进骨增重。我们的实验策略旨在证明这种潜在的合成代谢效应NET阻断，并表征这种作用的分子机制。在这项应用中，我们提出：1)表征发育和衰老过程中net -缺陷引起的细胞和结构骨异常，以确定net -缺陷小鼠的表型是由获得峰值骨量还是抵抗年龄相关的骨质流失引起的；2)利用体内和体外实验选择性地靶向每种细胞系，确定成骨细胞和神经元中Net缺乏对Net缺乏小鼠高骨量的相对贡献；3)表征选择性NET抑制剂对小鼠骨重塑、结构和力学性能的影响，以证明选择性NET阻滞剂可以促进骨增重的概念。公共卫生相关性：5 -羟色胺转运体家族的抗抑郁药占美国所有抗抑郁药使用量的62%，与骨质流失有关。我们的基因研究表明，另一类针对去甲肾上腺素转运体的抗抑郁药可能反而会促进骨骼生长。我们的目标是证明这些药物对小鼠骨骼的影响，并描述其中的分子机制。

DESCRIPTION (provided by applicant): The studies proposed in this application have the long-term goal to design a novel bone anabolic treatment. Anti-depressants, depending on their selectivity, target the serotonin transporter (SERT) or the norepinephrine transporter (NET) in neurons. The use of anti-depressants of the serotonin transporter family has been associated with bone loss and increased fracture risk. In contrast, anti-depressants of the NET family, in spite of having weak serotonin transporter selectivity, have not shown such a negative effect on bone and even appear to reduce fracture risk in one clinical study. Our preliminary data demonstrates that genetic blockade of NET in mice provokes a dramatic increase in bone volume, caused by a reduction in bone resorption and an increase in bone formation. Net is expressed in nerve cells where its function is to clear norepinephrine (NE) from the synapse, but surprisingly also in osteoblasts, where its function has been unexplored. We have demonstrated in our previous studies that NE released from sympathetic nerves, via the beta 2 adrenergic receptor (¿2AR), induces bone loss. Based on these studies, one would expect that mice lacking NET would be characterized by NE accumulation at the synapse and thus bone loss. The opposite phenotype we have found in Net-deficient mice led us to propose three possible mechanisms by which Net deficiency could cause a high bone mass: a) chronic ¿2AR stimulation in osteoblasts that leads to receptor desensitization and blockade of the anti-osteogenic signal from sympathetic nerves, b) low sympathetic tone caused by stimulation of inhibitory a2AR in the brain, and/or c) a cell-autonomous inhibitory function of NET on osteoblast differentiation or function. Regardless of the mechanism of action, our preliminary results identify NET as a potential molecular target for drug discovery of a novel class of bone anabolic agents and have led us to hypothesize that clinically available selective NET blockers could promote bone gain. Our experimental strategies aim at demonstrating this potential anabolic effect NET blockade and at characterizing the molecular mechanism(s) of action underlying this effect. In this application, we propose: 1) To characterize the cellular and structural bone abnormalities induced by Net-deficiency during development and aging, in order to determine whether the phenotype of Net-deficient mice is caused by the acquisition of a high peak bone mass or resistance to age-associated bone loss; 2) To determine the relative contribution of Net deficiency in osteoblasts and neurons to the high bone mass of Net-deficient mice, using both in vivo and in vitro assays to selectively target each cell lineage; 3) To characterize the effect of selective NET inhibitors on bone remodeling, architecture and mechanical properties in mice, as a proof of concept of the notion that selective NET blockers can promote bone gain. PUBLIC HEALTH RELEVANCE: Anti-depressants of the serotonin transporter family, accounting for 62% of all anti-depressant use in the US, has been associated with bone loss. Our genetic studies suggest that another class of anti-depressants, targeting the norepinephrine transporter, may instead promote bone gain. Our goal is to demonstrate the effect of these drugs on bone in mice and to characterize the molecular mechanisms in play.