Ribosome Dysfunction in Neurological Disorders

 描述(申请人提供)：神经退行性疾病影响世界各地数百万人，特别是在老龄化人口中。这些疾病中的绝大多数不是家族性的，与这些罕见的家族性疾病形式有关的突变突显了这一组疾病的复杂性。为了开始理解这种复杂性，我们使用了先进的遗传学方法来精确定位在老化的哺乳动物大脑中维持神经元动态平衡的分子途径。使用这种方法，我们最近证明了未解决的核糖体停滞是神经退变的一种新机制。尽管翻译的基本重要性，但在哺乳动物细胞中核糖体停滞的细胞后果一直是未知的，直到我们发现一种新的哺乳动物核糖体拯救因子Gtpbp2的突变导致小脑颗粒细胞、皮质和海马神经元以及多个视网膜神经元的共济失调和变性。重要的是，我们证明了Gtpbp2的丢失与广泛使用的C57BL6/J(B6J)小鼠品系中CNS特异的细胞质tRNAArgUCU中的突变发生上位性相互作用，导致神经退化。我们的核糖体足迹实验显示，这种tRNA的丢失导致精氨酸AGA密码子的低水平核糖体停滞，这与神经退化无关。然而，在没有Gtpbp2的情况下，停滞显著增加，这表明该蛋白通常可以解决核糖体停滞。在这项应用中，我们建议确定这些和其他核糖体拯救因子在神经元存活中的功能，年龄增加对脑中核糖体停滞的影响，以及导致哺乳动物神经元核糖体停滞的其他分子机制。在目标1中，我们将确定核糖体拯救因子Gtpbp1、Hbs1l和Pelo在tRNA缺乏和不缺乏的情况下丢失的影响。这些研究将得到新的计算方法的补充，以更精确地推断核糖体的位置，并利用翻译过程的参数依赖模拟来确定区分菌株的机制。在目标2中，我们将确定衰老对衰老野生型和无tRNA突变的核糖体拯救突变小鼠脑内核糖体停滞和神经变性的影响，并从老龄小鼠的小脑中产生和分析核糖体足迹和RNA-Seq数据。在目标3中，我们将研究导致细胞死亡的途径，并确定它们对神经元的独特性。我们将确定普遍表达的tRNAs的缺乏是否会导致核糖体停滞和其他器官的病理变化，分析GCN2/ATF4和P53通路对神经退变的影响，并在Gtpbp2-/-小鼠中寻找其他神经退变的修饰基因。总之，我们希望这些研究能够揭示翻译延长失调导致细胞死亡的机制及其对神经退行性疾病的特异性。

 DESCRIPTION (provided by applicant): Neurodegenerative disorders affect many millions of people around the world, particularly in the aging population. The vast majority of these diseases are not familial and the mutations that have been associated with rare familial forms of these disorders underscore the complexity of this group of diseases. To begin to understand this complexity, we have used forward genetic approaches to pinpoint the molecular pathways that maintain neuronal homeostasis in the aging mammalian brain. Using this approach we recently demonstrated that unresolved ribosome stalling is a novel mechanism for neurodegeneration. Despite the fundamental importance of translation, the cellular consequences of ribosome stalling in mammalian cells had been unknown until our discovery that a mutation in a novel mammalian ribosome rescue factor Gtpbp2 causes ataxia and degeneration of cerebellar granule cells, cortical and hippocampal neurons, and multiple retinal neurons. Importantly we demonstrated that loss of Gtpbp2 epistatically interacts with a mutation in a CNS- specific, cytoplasmic tRNAArgUCU in the widely used C57BL6/J (B6J) mouse strain to cause neurodegeneration. Our ribosome footprinting experiments revealed that loss of this tRNA led to low levels of ribosome stalling at Arginine AGA codons that was not associated with neurodegeneration. However, stalling was dramatically increased in the absence of Gtpbp2, demonstrating that this protein normally resolves ribosomal stalls. In this application we propose to determine the function of these and other ribosome rescue factors in neuron survival, the impact of increasing age on ribosome stalling in the brain, and additional molecular mechanisms which cause ribosome stalling in mammalian neurons. In Aim 1 we will determine the effects of loss of the ribosome rescue factors Gtpbp1, Hbs1l, and Pelo with- and without- tRNA deficiency. These studies will be complemented by novel computational methods to infer ribosomal locations at increased precision and ascertain mechanisms that distinguish strains using parameter-dependent simulations of the translation process. In Aim 2 we will determine the effects of aging on ribosome stalling and neurodegeneration in the brains of aged wild type and ribosome rescue mutant mice without the tRNA mutation and generate and analyze ribosome footprinting and RNA-Seq data from cerebella of aged mice. In Aim 3 we will investigate pathways that lead to cell death and determine their uniqueness for neurons. We will determine if deficiency of ubiquitously expressed tRNAs induces ribosome stalling and pathology in other organs, analyze the effects of the GCN2/ATF4 and P53 pathways on neurodegeneration, and identify additional modifier genes of neurodegeneration in Gtpbp2-/- mice. Together, we expect these studies to reveal the mechanisms by which dysregulation of translation elongation leads to cellular death and their specificity for neurodegenerative disease.