Exosomal protein deficiencies: how abnormal RNA metabolism results in childhood-onset neurological diseases

所有的基因都被复制成短寿命的RNA分子，然后被翻译成蛋白质，形成体内细胞的构建盒。DNA的转录、pre-mRNA加工成成熟mRNA（剪接）、mRNA的降解和翻译成蛋白质都受到严格的调控。某种RNA种类过多或过少对细胞都可能是危险的，但我们对微调RNA数量的机制的理解仍然有限。基因表达的调节（RNA代谢）对正常细胞功能至关重要，但神经细胞似乎无法应对错误。越来越多的婴儿或儿童严重的遗传性神经系统疾病是由RNA生产机制的缺陷引起的，这说明了这一点；这些疾病的特点是大脑和中枢神经系统的发育和/或结构异常。本提案的目的是研究和表征一种新型的RNA相关神经系统疾病，这种疾病是由一种称为人类外泌体的多蛋白复合物的功能障碍引起的。外泌体的作用包括RNA的降解和成熟。外显子亚基基因EXOSC3的突变在脑干和小脑的婴儿期变性和脊髓运动神经元功能障碍引起的肌肉无力患者中有报道。我们最近在患有类似严重神经系统疾病的婴儿中发现了EXOSC8的突变，该突变编码人类外泌体的另一个组成部分。此外，这些孩子还失去了大脑和脊髓中的髓磷脂，而髓磷脂通常是包裹和隔离神经的，并起到加速信号传递的作用。我们还在一名患有严重脊髓运动神经元功能障碍的儿童中发现了一种新基因RBM7的纯合突变。RBM7是人类外泌体的相互作用伙伴。这些儿童的严重和孤立的神经系统症状提出了外泌体在神经元中特别重要的可能性；然而，仍有许多悬而未决的问题。本提案的目的是进一步表征外泌体在神经细胞中的作用，并将解决以下几个方面：1。来自患者的神经细胞：我们将分析患者来源的皮肤细胞中的RNA水平，并将用最近发表的方法将其转化为神经细胞。我们将通过不同细胞类型的外泌体的变化来确定哪些RNA物种被修饰。2. 转基因斑马鱼：与此同时，我们将创建斑马鱼外泌体蛋白缺乏模型。通过人工去除单个外泌体成分并在斑马鱼胚胎中引入突变，我们将探索其对大脑和不同类型神经元的发育和功能的影响。此外，我们还将研究修饰外泌体对斑马鱼髓磷脂形成的影响。我们的研究结果将确定外泌体在调节基因表达中的最重要作用，以及为什么它对神经元的损害更大。结合人类细胞和斑马鱼的数据将使我们能够更好地定义外泌体的重要基因和相互作用的伙伴。通过修饰外泌体成分或一些本文确定的相互作用因子，我们可能会发现改变神经元中RNA降解或加工的潜在途径，这可以进一步发展为外泌体疾病或其他类型的由异常RNA功能引起的神经退行性疾病的治疗方法。

All genes are copied into short-lived RNA molecules, which are then translated to protein, forming the building box of the cells in the body. The transcription of DNA, the processing of pre-mRNA into mature mRNA (splicing), the degradation of mRNA and the translation into proteins are all tightly regulated. Both too much and too little of a certain RNA species could be dangerous for a cell, but our understanding of the mechanisms to fine-tune the RNA amounts is still limited. The regulation of gene expression (RNA metabolism) is of utmost importance for normal cell function, nerve cells, however, seem less able to cope with errors. This is illustrated by an increasing number of severe inherited neurological diseases of infancy or childhood caused by a defect in the RNA production machinery; these disorders are characterized by abnormalities of the development and/or the structure of the brain and the central nervous system. The aim of this proposal is to study and characterize a novel form of RNA related neurological disorder due to dysfunction of a multi-protein complex called the human exosome. The role of the exosome consists of degradation and maturation of RNA. Mutations in the exosome subunit gene EXOSC3 were reported in patients with infantile-onset degeneration of the brainstem and cerebellum and muscle weakness due to spinal motor neuron dysfunction. We have recently identified mutations in EXOSC8, encoding another component of the human exosome, in infants with a similar severe neurological disease. In addition, these children also lose myelin in the brain and spinal cord, which normally coats and insulates nerves and serves to speed up signalling. We also identified a homozygous mutation in a novel gene RBM7 in a child with severe spinal motor neuron dysfunction. RBM7 is an interacting partner of the human exosome. The severe and isolated neurological symptoms in these children raise the possibility that the exosome is particularly important in neurons; however, there are still many open questions. The aim of this proposal is to further characterize the role of the exosome in nerve cells and will address the following aspects:1. Neuronal cells from patients: We will analyse RNA levels in patient derived skin cells and will transform them into neuronal cells with recently published methods. We will determine which RNA species are modified by changes in the exosome in the different cell types. 2. Genetically modified zebrafish: In parallel, we will create zebrafish models of exosomal protein deficiencies. By artificially removing single exosome components and by introducing mutations in zebrafish embryos we will explore its effect on the development and function of the brain and different types of neurons. In addition, we will also study the effect of modifying the exosome on myelin formation in zebrafish. Our results will identify the most important roles of the exosome in regulating gene expression and why is it more damaging for neurons. Combining the data in human cells and zebrafish will enable us to better define the important genes and interacting partners of the exosome. By modifying exosome components or some of the here identified interacting factors we may discover potential pathways to alter RNA degradation or processing in neurons, which can be further developed as a therapeutic approach in exosomal diseases or in other types of neurodegenerative diseases caused by abnormal RNA function.