Novel mechanistic study of CMT2D neuropathy

Charcot Marie Tooth 2D （CMT2D）是由甘氨酸RNA合成酶基因（GlyRS）显性突变引起的。尽管普遍需要这种细胞质酶来生成甘氨酸- trna进行翻译，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CMT2D最初被认为是由GlyRS酶不足引起的，但先前的遗传学研究表明，CMT2D是由GlyRS的新形态功能（即功能获得）引起的。突变GlyRS引起CMT2D的作用机制尚不清楚，因此是本研究的重点。我们的合作者杨向磊实验室的初步体外和生化数据揭示了一个迄今未知的生物学途径，其中GlyRS分泌并与VEGF竞争性地结合到Nrp1受体。由于运动神经元表达Nrp1，而VEGF异构体164具有神经营养活性，他们假设突变GlyRS和Nrp1之间新的细胞外相互作用是CMT2D的基础。这项探索性R21资助的目的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CMT2D疾病的治疗剂。四个目的是(1)确定小鼠是否分泌GlyRS（正常和突变蛋白），(2)表征CMT2D小鼠的运动缺陷，并确定GlyRS是否与VEGF和Nrp1在体内遗传相互作用，(3)检测GlyRS对Nrp1信号的影响，(4)使用AAV-VEGF载体在CMT2D小鼠肌肉中表达VEGF，并检测周围神经病变（即轴突损失）的进展是否被减缓或阻止。这些研究可能会导致我们对正常GlyRS在广泛的生物学背景下作为细胞外配体功能的理解的范式转变，但这里的主要目标是确定突变GlyRS是否获得了作为运动神经元Nrp1信号的VEGF拮抗剂的新形态活性。该资助旨在建立足够的初步数据，以证明VEGF作为CMT2D可行的新治疗方法的进一步研究。

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.