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周围神经系统的轴突变性。甘酰基tRNA合成酶（GARS）和酪氨酸tRNA合成酶（YARS）突变分别导致Charcot-Marie-Tooth型D和显性中间Charcot-Marie-Tooth型C （CMT2D和DI-CMTC）。这些酶具有将氨基酸装载到同源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。我们已经用CMT2D小鼠模型证明了这一点，在CMT2D小鼠模型中，尽管突变蛋白的酶活性正常，但GARS中的氨基酸替代会导致显性周围神经病变，而消除GARS表达的功能缺失等位基因不会导致显性表型，尽管酶活性总体水平较低。这些结果使我们假设突变形式的GARS通过未知的非规范功能的丧失或通过病理性的功能获得导致疾病。因此，我们提出了三个目标，以更好地了解CMT2D和DI-CMTC的发病机制。在Aim 1中，我们将使用“条件敲入”策略将人类疾病等位基因引入小鼠Yars基因中，建立小鼠DI-CMTC模型。初步研究表明，将Yars的表达降低到野生型水平的35%不会引起周围神经病变，这表明像Gars一样，Yars蛋白的突变形式是导致疾病的必要条件。Yars条件敲入小鼠将使我们进一步探索GARS和Yars突变是否确实具有共同的致病机制。在目标2中，我们将测试一种基于体外和生化数据的疾病机制。Schimmel和Yang。该模型提出，GARS和DAXX（一种涉及其他运动神经元疾病途径的蛋白质）之间相互作用的改变可能是CMT2D的基础。我们将在体内测试这一点，再次使用条件敲入策略靶向GARS中对DAXX相互作用至关重要的残基。如果提出的机制是正确的，这应该导致严重的神经病变表型。在Aim 3中，我们将结合小鼠和人类遗传学来了解CMT2D不同严重程度的遗传基础。CMT2D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。我们能够在小鼠中复制这种变异，并在Chr. 1上绘制了一个位点，该位点有助于保存感觉轴突。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导致CMT2D患者的不同严重程度。修饰位点的鉴定将提高我们对CMT2D机制的理解，提出治疗策略，并提高CMT2D患者的诊断和预后。

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).