Novel mechanistic study of CMT2D neuropathy

Charcot玛丽牙2D（CMT 2D）是由甘氨酸RNA合成酶基因（GlyRS）的显性突变引起的。尽管这种细胞质酶在产生用于翻译的甘氨酸-tRNA时普遍需要，但突变会导致选择性外周神经元轴突变性，导致运动和感觉缺陷。虽然CMT 2D最初被认为是由GlyRS酶不足引起的，但先前的遗传研究表明，CMT 2D是由GlyRS的新变体功能（即功能获得）引起的。突变体GlyRS导致CMT 2D的作用机制尚不清楚，因此是该补助金的重点。来自我们的合作者Xiang-Lei Yang实验室的初步体外和生化数据揭示了一种迄今未知的生物学途径，其中GlyRS被分泌并与VEGF竞争性结合到Nrp 1受体。由于运动神经元表达Nrp 1，VEGF亚型164具有神经营养活性，他们假设突变体GlyRS和Nrp 1之间的新型细胞外相互作用支持CMT 2D。这项探索性R21资助的目标是确定他们假设的关键方面是否正确，以确定VEGF是否可以用作CMT 2D疾病的治疗药物。四个目标是：（1）确定GlyRS是否（正常和突变蛋白）在小鼠中分泌，（2）表征CMT 2D小鼠中的运动缺陷并确定GlyRS是否在体内与VEGF和Nrp 1遗传相互作用，（3）测定GlyRS对Nrp 1信号传导的影响，和（4）使用AAV-VEGF载体在CMT 2D小鼠肌肉中表达VEGF，并测定周围神经病变的进展（即轴突损失）是否被减缓或阻止。这些研究可能会导致一个范式的转变，在我们的理解正常的GlyRS功能作为一个细胞外配体在广泛的生物背景下，但这里的主要目标是确定突变GlyRS是否已获得了neomorphic活动作为VEGF拮抗剂的Nrp 1信号在运动神经元。该基金旨在建立足够的初步数据，以证明进一步研究VEGF作为CMT 2D的可行和新颖的治疗方法。

Charcot Marie Tooth 2D (CMT2D) is caused by dominant mutations in the glycine RNA synthetase gene (GlyRS). Despite the ubiquitous requirement for this cytoplasmic enzyme in generating glycine-tRNA for translation, mutations cause selective peripheral neuron axon degeneration leading to motor and sensory deficits. Although CMT2D was initially thought to be caused by inadequate GlyRS enzyme, previous genetic studies indicate that CMT2D is caused by a neomorphic function of GlyRS (i.e. gain-of-function). The mechanism of action by which mutant GlyRS causes CMT2D is not known and therefore is the focus of this grant. Preliminary in vitro and biochemical data from our collaborator Xiang-Lei Yang's lab has unmasked a heretofore unknown biological pathway in which GlyRS is secreted and binds competitively with VEGF to the Nrp1 receptor. Since motor neurons express Nrp1, and VEGF isoform 164 has neurotrophin-activity, they hypothesize that the novel extracellular interaction between mutant GlyRS and Nrp1 underpins CMT2D. The goal in this exploratory R21 grant is to establish whether key aspects of their hypothesis are correct in order to determine if VEGF can be used as a therapeutic agent for CMT2D disease. The four aims are (1) to establish whether GlyRS (normal and mutant protein) is secreted in mice, (2) to characterize the motor deficits in CMT2D mice and determine if GlyRS genetically interacts with VEGF and Nrp1 in vivo, (3) to assay the influence of GlyRS on Nrp1 signaling, and (4) to express VEGF in CMT2D mouse muscles using AAV-VEGF vectors and assay whether the progression of peripheral neuropathy (i.e. axon loss) is slowed or prevented. These studies may lead to a paradigm shift in our understanding of normal GlyRS function as an extracellular ligand in a broad array of biological contexts, but the main goal here is to determine whether mutant GlyRS has acquired a neomorphic activity as a VEGF antagonist of Nrp1 signaling in motor neurons. This grant aims to establish sufficient preliminary data to justify further investigation of VEGF as a feasible and novel treatment for CMT2D.