Elucidating mitochondrial pathomechanisms of ENT3 disorders in ENT3 KO mice

描述（由申请方提供）：人平衡型核苷转运蛋白-3（hENT 3）（一种主要位于线粒体中的核苷转运蛋白）中的21个排他性突变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、皮肤增生、拇趾外翻、身材矮小等）。虽然hENT 3谱疾病和线粒体疾病之间存在有趣的相似性，但hENT 3在这些疾病的起始、进展和可能的干预中的机制参与尚不清楚。我们的长期目标是确定hENT 3谱系疾病的分子发病机制。本R 03资助申请的总体目标是证明/反驳线粒体生理学异常导致hENT 3谱系疾病的概念。具体来说，这是我们的中心假设，干扰线粒体核苷转运是hENT 3谱疾病的生理，生化和临床表现的分子解释。这一假设是基于初步数据，这些数据显示所有hENT 3疾病突变严重损害核苷的线粒体转运、线粒体定位和/或hENT 3蛋白的稳定性。提出的研究的基本原理是，在小鼠中证明线粒体核苷转运功能障碍是hENT 3谱疾病的根本原因，这将提供一个实验模型，随后深入研究这些疾病的分子发病机制和潜在的补救措施。这一中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ENT 3在线粒体路径生理学中的作用。工作假设是ENT 3将显著影响体外和体内小鼠线粒体功能。该假设是基于我们自己的初步数据，这些数据确定了hENT 3谱系综合征中线粒体核苷转运的减少。将使用源自人类疾病患者和mENT 3 KO小鼠的细胞来评价线粒体路径生理学。具体目标2将确定mENT 3 KO小鼠中病理变化的发生和挽救。工作假设是，在mENT 3 KO小鼠中发现的异常将密切模拟在hENT 3疾病中观察到的异常，并且线粒体转运功能的恢复将有助于逆转疾病病理学。该假说是基于hENT 3和线粒体疾病的临床表现之间的相似性和mENT 3发育表达的空间同步性。hENT 3病症表现与那些表现的小鼠等效物的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，并测试干扰线粒体核苷转运可能导致hENT 3疾病中观察到的异常类型的概念。这一贡献将是显着的，因为由此产生的模型将使随后的机制调查。拟议的研究是创新的，因为它将使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.