Elucidating mitochondrial pathomechanisms of ENT3 disorders in ENT3 KO mice

描述(申请人提供)：21人类平衡核苷转运体3(HENT3)的独有突变，这是一种主要位于线粒体的核苷转运体，可导致一系列人类遗传性疾病，具有广泛的皮肤和肌肉骨骼疾病表现(例如硬皮病、多毛症、拇指外翻、身材矮小等)。尽管hENT3谱系疾病和线粒体疾病之间有有趣的相似之处，但hENT3参与这些疾病的启动、进展以及干预的机制尚不清楚。我们的长期目标是确定hENT3谱系疾病的分子发病机制。这项R03拨款申请的总体目标是证明/反驳线粒体生理异常是hENT3谱系障碍的原因这一概念。具体地说，我们的中心假设是干扰线粒体核苷运输是hENT3谱系疾病的生理、生化和临床表现的分子解释。这一假说建立在初步数据的基础上，这些数据表明，所有hENT3疾病突变都严重损害线粒体核苷的运输、线粒体定位和/或hENT3蛋白的稳定性。这项拟议研究的基本原理是，在小鼠身上证明线粒体核苷转运功能障碍是hENT3谱系疾病的根本原因，这将为随后深入研究这些疾病的分子发病机制和潜在的治疗方法提供一个实验模型。这一核心假设将通过追求两个具体目标来检验。具体目标1将确定ENT3在线粒体路径生理学中的作用。工作假设是，ENT3将显著影响体外和体内小鼠线粒体功能。这一假说是基于我们自己的初步数据，这些数据确定了hENT3谱系综合征的线粒体核苷转运减少。来自人类疾病患者和mENT3 KO小鼠的细胞将被用于评估线粒体路径生理学。特异靶2将确定mENT3KO小鼠病理改变的发生和挽救。工作假说是，在mENT3 KO小鼠中发现的异常将非常类似于hENT3疾病中看到的异常，线粒体运输功能的恢复将有助于逆转疾病病理。这一假说是基于hENT3的临床表现与线粒体疾病之间的相似性以及mENT3发育表达的空间同步性。将hENT3紊乱症状与那些症状的老鼠等价物进行比较，将成为小鼠模型的特征。我们的研究将验证一个关键的疾病模型，并测试对线粒体核苷运输的干扰可能导致hENT3疾病中所见的异常类型的概念。这一贡献将是重大的，因为所产生的模式将使随后的机械性调查成为可能。这项拟议的研究具有创新性，因为它将使其适用于多种皮肤和肌肉骨骼疾病。

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.