Modelling the Role of Mitochondrial Aspartyl-tRNA Synthetase (DARS2) in Neurodegeneration

线粒体功能障碍，无论是原发性还是继发性，越来越多地被认为是神经退行性变的一个中心特征。这些疾病在我们老龄化人口中的日益普遍意味着了解线粒体参与神经元损失背后的机制是一个紧迫的公共卫生问题。近年来，常染色体隐性白质脑病伴脑干和脊髓受累和乳酸中毒综合征（LBSL）被添加到一大组线粒体疾病中。这种疾病的临床特征是缓慢进行性小脑共济失调、痉挛状态和背柱功能障碍，是由编码线粒体乙酰tRNA合成酶的DARS 2基因突变引起的。值得注意的是，其他线粒体tRNA合成酶基因的突变会导致具有非常组织特异性模式的疾病，尽管所有这些基因的主要功能都是在线粒体内的蛋白质合成中。 我们的项目将采用创新和新颖的细胞和动物模型来阐明DARS 2突变引起的神经退行性变的机制，并利用这些发现来破译线粒体疾病组织特异性的可能作用。我们将集中精力回答以下具体问题：（i）与前脑神经元和白色物质中DARS 2缺乏相关的细胞和组织发病机制是什么？(ii)DARS 2缺陷影响体内线粒体能力的机制是什么？(iii)神经元对LBSL患者中发现的特定DARS 2错义突变的高度敏感性的基础是什么？我们将通过分析前脑神经元中的DARS 2缺陷和我们已经产生的白色物质特异性敲除小鼠模型来解决这些问题。我们还将介绍在不同的神经元，神经和非神经细胞系中发现的最常见的错义突变，并分析它们对线粒体和细胞功能的影响。最后，我们建议使用新的CRISPR/Cas9技术来生成携带患者中发现的最常见的DARS 2错义突变的小鼠模型，以最准确地在体内模拟LBSL。我们相信，将动物模型与不同的哺乳动物细胞相结合的综合方法将对理解线粒体功能障碍在神经退行性变中的作用产生更大的影响。

Mitochondrial dysfunction, whether primary or secondary, is increasingly recognized as a central feature of neurodegeneration. The increasing prevalence of these diseases in our ageing populations means that understanding the mechanisms behind mitochondrial involvement in neuronal loss is a pressing public health issue. Recently, the autosomal recesive leucencephalopathy with brainstem and spinal cord involvement and lactacidosis syndrome (LBSL) is added to a large group of mitochondrial disorders. The disorder, clinically characterized by slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, is caused by mutations in the DARS2 gene, which encodes for the mitochondrial aspartyl tRNA synthetase. Remarkably, mutations in other mitochondrial tRNA synthetase genes give rise to disorders with a very tissue specific pattern, despite primary function of all these genes in the protein synthesis within mitochondria. Our project will employ innovative and novel cell and animal models to elucidate the mechanisms of neurodegeneration caused by DARS2 mutations and use the findings to decipher a possible role of tissue specificity of mitochondrial diseases. We will focus our efforts to answer specific questions: (i) What is the cellular and tissue pathogenesis associated with DARS2 deficiency in forebrain neurons and white matter? (ii) What are the mechanisms whereby DARS2 deficiency impacts mitochondrial proficiency in vivo? (iii) What is the basis for the exquisite sensitivity of neurons to specific DARS2 missense mutations found in LBSL patients? We will address these questions by analysing DARS2 deficiency in forebrain neurons and white matter specific knockout mouse models we already produced. We will also introduce the most common missense mutations found in patients in different neuronal, neural and non-neural cell lines and analyze their impact in on mitochondrial and cellular function. Finally, we propose to use novel CRISPR/Cas9 technology to generate a mouse model carrying the most common DARS2 missense mutation found in patients to most accurately model the LBSL in vivo. We believe that an integrative approach combining animal models with different mammalian cells will make a greater impact on the understanding of the role of mitochondrial dysfunction in neurodegeneration.