Elucidating mitochondrial pathomechanisms of ENT3 disorders in ENT3 KO mice

描述（由申请人提供）：人类平衡核苷转运体-3 （hENT3）的21个排他突变，主要定位于线粒体的核苷转运体导致一系列人类遗传疾病，具有广泛的皮肤和肌肉骨骼疾病表现（例如硬皮病，多毛症，外翻，身材矮小等）。尽管在hENT3谱系疾病和线粒体疾病之间存在有趣的相似之处，但hENT3在这些疾病的发生、进展和干预中的机制参与尚不清楚。我们的长期目标是确定hENT3谱系障碍的分子发病机制。这项R03拨款申请的总体目标是证明/反驳线粒体生理异常导致hENT3谱系障碍的概念。具体来说，我们的中心假设是，干扰线粒体核苷运输是hent3谱系障碍的生理、生化和临床表现的分子解释。这一假设基于初步数据，这些数据表明，所有hENT3疾病突变严重损害核苷的线粒体运输、线粒体定位和/或hENT3蛋白的稳定性。拟议研究的基本原理是，在小鼠中证明线粒体核苷转运功能障碍是hent3谱系障碍的根本原因，将为随后深入研究这些疾病的分子发病机制和潜在治疗方法提供一个实验模型。这一中心假设将通过追求两个具体目标来检验。特异性目的1将确定ENT3在线粒体路径生理学中的作用。工作假设是ENT3会显著影响小鼠体内和体外线粒体功能。该假设是基于我们自己的初步数据，这些数据确定了hENT3谱系综合征中线粒体核苷转运的减少。来自人类疾病患者和mENT3 KO小鼠的细胞将用于评估线粒体路径生理学。特异性靶2将决定ment3ko小鼠病理改变的发生和恢复。目前的假设是，在mENT3 KO小鼠中发现的异常与在hENT3疾病中发现的异常非常相似，线粒体运输功能的恢复将有助于逆转疾病病理。该假说基于hENT3的临床表现与线粒体疾病的相似性以及mENT3发育表达的空间同步性。将hENT3紊乱表现与小鼠等效的这些表现进行比较将表征小鼠模型。我们的研究将验证一个关键的疾病模型，并验证干扰线粒体核苷运输可能导致hENT3疾病中出现的异常类型的概念。这一贡献将是重要的，因为由此产生的模型将使后续的机制研究成为可能。拟议的研究是创新的，因为它将使多种皮肤和肌肉骨骼疾病的适用性。

DESCRIPTION (provided by applicant): Twenty one exclusive mutations in the human equilibrative nucleoside transporter-3 (hENT3), a nucleoside transporter predominantly localized in mitochondria cause a spectrum of human genetic disorders with wide-ranging skin and musculoskeletal disease manifestations (e.g. scleroderma, hypertrichosis, hallus valgus, short stature, etc.). Although there are intriguing similarities between the hENT3-spectrum disorders and mitochondrial disorders, the mechanistic involvement of hENT3 in the initiation, progression, and perhaps in the intervention of these disorders is not understood. Our long-term goal is to identify the molecular pathogeneses of hENT3 spectrum disorders. The overall objective of this R03 grant application is to prove/disprove the concept that abnormalities of mitochondrial physiology are responsible for hENT3 spectrum disorders. Specifically, it is our central hypothesis that interference with mitochondrial nucleoside transport is the molecular explanation for the physiological, biochemical, and clinical manifestations of hENT3-spectrum disorders. This hypothesis is based on preliminary data which show that all hENT3 disease mutations severely impair mitochondrial transport of nucleosides, mitochondrial localization, and/or the stability of hENT3 protein. The rationale underlying the proposed research is that proof in mice that dysfunctional mitochondrial nucleoside transport is the root cause of hENT3-spectrum disorders would provide an experimental model in which to subsequently investigate, in depth, the molecular pathogeneses and potential remedies for these diseases. This central hypothesis will be tested by pursuing two specific aims. Specific aim 1 will determine the role of ENT3 in mitochondrial path physiology. The working hypothesis is that ENT3 will significantly influence in vitro and vivo mouse mito- chondrial functions. The hypothesis is based on our own preliminary data which identify the reduction of mito- chondrial nucleoside transport in hENT3 spectrum syndromes. Cells derived from human disease patients and mENT3 KO mice will be utilized to evaluate mitochondrial path physiology. Specific aim 2 will determine the occurrence and rescue of pathologic changes in mENT3 KO mice. The working hypothesis is that the abnormalities discovered in mENT3 KO mice will closely mimic those seen in hENT3 disorders and that the restoration of mitochondrial transport functions will help in reversal of disease pathology. The hypothesis is based on the similarities between the clinical manifestations of hENT3 and mitochondrial disorders and on the spatial synchrony of mENT3 developmental expression. Comparisons of hENT3 disorder manifestations with the mouse-equivalent of those manifestations will characterize the mouse model. Our studies will validate a crucial disease model and test the concept that interference with mitochondrial nucleoside transport could result in the types of abnormalities seen in hENT3 disorders. This contribution would be significant because the resultant model would enable subsequent mechanistic investigations. The proposed research is innovative because it would enable applicability to multiple skin and musculoskeletal disorders.