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疗法。抗抑郁药根据其选择性，靶向神经元中的5-羟色胺转运体(SERT)或去甲肾上腺素转运体(NET)。5-羟色胺转运体家族的抗抑郁药的使用与骨质丢失和增加骨折风险有关。相比之下，Net家族的抗抑郁药尽管对5-羟色胺转运体的选择性很弱，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。我们的初步数据显示，在小鼠中，Net的基因阻断引起骨体积的急剧增加，这是由于骨吸收减少和骨形成增加所致。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，它也表达在成骨细胞中，其功能尚未被探索。我们在以前的研究中已经证明，交感神经通过β2肾上腺素能受体(β2 AR)释放去甲肾上腺素，导致骨丢失。基于这些研究，人们预计缺乏Net的小鼠的特征是突触处去甲肾上腺素积聚，从而导致骨丢失。我们在Net缺陷小鼠中发现的相反表型导致我们提出了三种可能的机制，通过Net缺乏可能导致高骨量：a)成骨细胞的慢性2AR刺激导致交感神经受体脱敏和反成骨信号的阻断，b)刺激脑中抑制性a2AR导致交感神经张力降低，和/或c)Net对成骨细胞分化或功能的细胞自主抑制功能。不管作用机制如何，我们的初步结果确定Net是一种新型骨合成代谢药物的潜在分子靶点，并使我们假设临床上可用的选择性Net阻滞剂可以促进骨生长。我们的实验策略旨在展示这种潜在的合成代谢效应，并描述这种作用的分子机制(S)。在这一应用中，我们建议：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于获得高峰值骨量还是对年龄相关性骨丢失的抵抗；2)通过体内和体外实验，选择性地针对每个细胞系，确定Net缺陷小鼠成骨细胞和神经元中Net缺陷对高骨量的相对贡献；3)表征选择性网络阻滞剂对小鼠骨改建、结构和力学性能的影响，作为选择性网络阻滞剂可以促进骨生长的概念的证据。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的62%，与骨质丢失有关。我们的遗传学研究表明，另一类针对去甲肾上腺素转运体的抗抑郁药物可能反而会促进骨骼增加。我们的目标是证明这些药物对小鼠骨骼的影响，并表征发挥作用的分子机制。

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.