Genetics of the Neuromuscular Junction: Mechanisms and Disease Models

描述（由申请人提供）：本提案的目的是了解tRNA合成酶基因的显性突变如何导致外周神经系统的轴突变性。甘氨酰tRNA合成酶（加尔斯）和酪氨酰tRNA合成酶（YARS）的突变分别导致Charcot-Marie-Tooth D型和显性中间Charcot-Marie- Tooth C型（CMT 2D和DI-CMTC）。这些酶的特征在于其将氨基酸装载到其同源tRNA上进行翻译的功能。然而，这些突变对这种活性的影响与疾病表型之间几乎没有相关性。我们已经使用CMT 2D小鼠模型证明了这一点，其中加尔斯中的氨基酸取代导致显性周围神经病变，尽管突变蛋白具有正常的酶活性，而消除加尔斯表达的功能丧失等位基因不会导致显性表型，尽管酶活性的总体水平较低。这些结果使我们推测，突变形式的加尔斯引起的疾病，通过一个未知的，非典型的功能，或通过一个病理性的功能获得的损失。因此，我们提出了三个目标，以更好地了解CMT 2D和DI-CMTC的疾病机制。在目标1中，我们将使用“条件性敲入”策略将人类疾病等位基因引入小鼠Yars基因来创建DI-CMTC小鼠模型。初步研究表明，将Yars表达降低至野生型水平的35%不会引起周围神经病变，这表明像加尔斯一样，需要YARS蛋白的突变形式才能引起疾病。Yars条件性基因敲入小鼠将使我们能够进一步探索加尔斯和YARS突变是否确实具有共同的致病机制。在目标2中，我们将根据我们的合作者Schimmel和Yang博士的体外和生化数据测试拟议的疾病机制。该模型提出，加尔斯和DAXX（一种与其他运动神经元疾病通路有关的蛋白质）之间的相互作用改变可能是CMT 2D的基础。我们将通过再次使用条件性敲入策略靶向加尔斯中对DAXX相互作用至关重要的残基来在体内测试这一点。如果提出的机制是正确的，这应该会导致严重的神经病变表型。在目标3中，我们将结合联合收割机小鼠和人类遗传学，以了解CMT 2D不同严重程度的遗传基础。CMT 2D患者可能表现为纯粹的运动缺陷（诊断为远端脊髓性肌萎缩症V型，dSMAV），而其他携带相同突变的患者则表现为运动和感觉缺陷。我们能够在小鼠中重现这种变异性，并在Chr. 1上绘制了一个有助于保存感觉轴突的位点。我们将使用小鼠遗传学来鉴定Chr. 1上的修饰基因。我们还将与Albena Jordanova博士合作，确定人类基因组的同一区域是否有助于CMT 2D患者的不同严重程度。修饰基因位点的鉴定将提高我们对CMT 2D机制的理解，提出治疗策略，并提高CMT 2D患者的诊断/预后。 公共卫生相关性：该提案旨在将外周神经系统遗传性疾病（腓骨肌萎缩症）小鼠模型的联合收割机研究与人类遗传学研究结合起来，以更好地了解疾病机制并提出新的治疗策略。这项工作将改善人类的遗传诊断，为临床前治疗测试创建直接相关的动物模型，并为这些疾病以及可能的其他运动神经元疾病（如肌萎缩侧索硬化症）确定新的治疗靶点。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand how dominant mutations in tRNA synthetase genes cause axonal degeneration in the peripheral nervous system. Mutations in glycyl tRNA synthetase (GARS) and tyrosyl tRNA synthetase (YARS) cause Charcot-Marie-Tooth type D and Dominant Intermediate Charcot-Marie- Tooth type C (CMT2D and DI-CMTC) respectively. These enzymes are well characterized for their function in charging amino acids onto their cognate tRNAs for translation. However, there are few correlations between the effects of these mutations on this activity and the disease phenotypes. We have demonstrated this using a mouse model of CMT2D, in which an amino acid substitution in GARS causes a dominant peripheral neuropathy despite normal enzymatic activity of the mutant protein, whereas a loss-of-function allele that eliminates Gars expression does not cause a dominant phenotype despite lower overall levels of enzyme activity. These results lead us to hypothesize that mutant forms of GARS cause disease either through the loss of an unknown, noncanonical function, or through a pathological gain-of-function. We therefore propose three aims to better understand the disease mechanisms of CMT2D and DI-CMTC. In Aim 1, we will create a mouse model of DI-CMTC using a "Conditional knockin" strategy to introduce human disease alleles into the mouse Yars gene. Preliminary studies indicate that decreasing Yars expression to 35% of wild type levels does not cause a peripheral neuropathy, suggesting that like Gars, a mutant form of the YARS protein is required to cause disease. The Yars conditional knockin mice will allow us to further explore whether GARS and YARS mutations do indeed share a common pathogenic mechanism. In Aim 2, we will test a proposed disease mechanism based on in vitro and biochemical data from our collaborators, Drs. Schimmel and Yang. This model proposes that altered interaction between GARS and DAXX, a protein implicated in other motor neuron disease pathways, may underlie CMT2D. We will test this in vivo by again using a conditional knockin strategy to target residues in GARS that are essential for the DAXX interaction. If the proposed mechanism is correct, this should cause a severe neuropathy phenotype. In Aim 3, we will combine mouse and human genetics to understand the genetic basis for the variable severity of CMT2D. CMT2D patients may present with purely motor deficits (diagnosed as distal Spinal Muscular Atrophy type V, dSMAV), whereas others carrying the same mutation present with both motor and sensory deficits. We are able to reproduce this variability in mice and have mapped a locus on Chr. 1 that contributes to the preservation of sensory axons. We will use mouse genetics to identify the modifying gene on Chr. 1. We will also determine if the same region of the human genome contributes to variable severity in CMT2D patients in collaboration with Dr. Albena Jordanova. The identification of modifier loci will improve our understanding of CMT2D mechanisms, suggest therapeutic strategies, and enhance diagnosis/prognosis of CMT2D patients. PUBLIC HEALTH RELEVANCE: This proposal seeks to combine research in mouse models of hereditary diseases of the peripheral nervous system (Charcot-Marie-Tooth Disease) with research in human genetics to better understand disease mechanisms and to suggest new therapeutic strategies. This work will lead to improved genetic diagnoses in humans, create directly relevant animal models for preclinical testing of therapies, and identify new therapeutic targets for these diseases, and possibly other motor neuron diseases such as Amyotrophic Lateral Sclerosis (ALS).