Investigating RNA dysregulation in Neurological Disease through study of Pontocerebellar Hypoplasia Type 1b

通过 1b 型桥小脑发育不全研究来调查神经系统疾病中的 RNA 失调

• 批准号: 10638196
• 负责人: Derrick Morton
• 金额: $ 42.84万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638196
• 关键词: Affect; Alleles; Amino Acid Substitution; Amino Acids; Atrophic; Attention; Behavior; Biochemical; Biological; Biological Models; Biology; Biophysics; Brain; Brain region; Breathing; CCRL2 gene; CRISPR/Cas technology; Catalytic Domain; Cell Nucleus; Cell model; Cells; Cerebellum; Child; Childhood; Clinical; Complement; Complex; Cytoplasm; Data; Defect; Development; Developmental Delay Disorders; Disease; Drosophila genus; Enhancers; Etiology; Fishes; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Genomics; Genotype; Goals; Heterogeneity; Homeostasis; Homologous Gene; Human; Impairment; In Situ; Individual; Inherited; Life; Link; Longevity; Mass Spectrum Analysis; Methods; Microcephaly; Missense Mutation; Modeling; Molecular; Molecular Machines; Motor; Movement; Muscular Atrophy; Mutation; Nervous System; Neurodevelopmental Disorder; Neuronal Dysfunction; Neurons; Pancreatic ribonuclease; Pathology; Phenotype; Play; Pontine structure; Pontocerebellar hypoplasia; Post-Transcriptional RNA Processing; Post-Transcriptional Regulation; RNA; RNA Decay; RNA Processing; Reporting; Ribonucleases; Role; Series; Severities; Spinal Cord; System; Technology; Testing; Tissues; Transcript; Variant; Work; autosome; behavioral phenotyping; brain morphology; dosage; exosome; fly; in vivo; in vivo Model; insight; motor behavior; motor learning; mutant; nervous system disorder; neurodevelopment; novel; palliative; sensory system; single-cell RNA sequencing; tool; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY/ABSTRACT Pontocerebellar Hypoplasia Type 1b (PCH1b) is an autosomal recessive neurological disorder characterized by hypoplasia/atrophy of the cerebellum and pons that is often fatal within the first year of life. The cerebellum and pons integrate information from sensory systems, the spinal cord, and other parts of the brain to regulate motor movements, breathing, and learning motor behavior. Individuals with PCH1b show muscle atrophy/weakness, microcephaly, and developmental delay. Most individuals with PCH1b do not live past childhood and current treatment is purely palliative. Mutations that cause PCH1b occur in the EXOSC3 gene, which encodes a structural cap subunit of an evolutionarily conserved and ubiquitously expressed RNA processing complex, the RNA exosome. The RNA exosome is a ribonuclease composed of both structural and catalytic subunits that play a critical role in the post-transcriptional regulation of RNA. This complex is required for 3’ to 5’ processing and degradation of a vast number of RNAs in both the nucleus and cytoplasm. Post-transcriptional processing of RNA is a critical regulatory step in gene expression, as underscored by the number of neurological diseases caused by defects in RNA processing factors. The tissue-specific phenotypes caused by the RNA exosome complex are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. Thus, we aim to investigate the in vivo functional consequences of distinct disease-causing amino acid substitutions in EXOSC3 that are linked to a range of mild to severe phenotypes in PCH1b. We have generated an allelic series of EXOSC3 disease-linked missense mutations in the Drosophila orthologue Rrp40 via CRISPR/Cas9 editing technology. Our previous work in flies revealed an enhanced requirement for Rrp40 in neurons. Furthermore, our RNA-seq analysis of brain-enriched transcriptomes of Rrp40 mutants revealed increases in steady-state levels of functionally important neuronal transcripts, suggesting that disease-causing amino acid changes in the Drosophila RNA exosome subunit Rrp40 contribute to neuronal dysfunction. Our goal now is to characterize how disease-causing amino acid substitutions in Rrp40 alter the molecular and cellular landscape of the developing nervous system in Drosophila in vivo. We will test the hypothesis that the RNA exosome regulates RNAs that are critical for proper neurodevelopment and function, a distinct subset of which are regulated by the RNA exosome cap subunit Rrp40 (EXOSC3) through three complementary aims: 1) Assess the functional consequences of amino acid substitutions in the Drosophila RNA exosome subunit Rrp40 corresponding to those that cause PCH1b; 2) Interrogate how Rrp40 mutations affect expression and localization of key neuronal transcripts within the fly brain; and 3) Exploit a genetic screen to determine whether aberrant accumulation of specific RNA exosome targets disrupt fly development and/or homeostasis. Successful completion on these aims will provide a synergistic understanding of RNA exosome biology and invaluable insights into PCH1b disease etiology.

项目概要/摘要 桥小脑发育不全 1b 型 (PCH1b) 是一种常染色体隐性神经系统疾病，其特征为 小脑和脑桥发育不全/萎缩，通常在出生后第一年内致命。小脑和 脑桥整合来自感觉系统、脊髓和大脑其他部分的信息来调节运动 动作、呼吸和学习运动行为。患有 PCH1b 的个体表现出肌肉萎缩/无力， 小头畸形和发育迟缓。大多数 PCH1b 患者无法活过童年和现在 治疗纯粹是姑息治疗。导致 PCH1b 的突变发生在 EXOSC3 基因中，该基因编码 进化上保守且普遍表达的RNA加工复合物的结构帽亚基， RNA外泌体。 RNA 外泌体是一种核糖核酸酶，由结构亚基和催化亚基组成，发挥作用 在RNA转录后调控中发挥着重要作用。该复合物是 3' 至 5' 处理所必需的，并且 细胞核和细胞质中大量 RNA 的降解。转录后加工 正如许多神经系统疾病所强调的那样，RNA 是基因表达中的关键调节步骤 由RNA加工因子缺陷引起。 RNA外泌体引起的组织特异性表型 根据当前的 RNA 外泌体功能模型，复杂的理解具有挑战性，且其功能有限 在任何体内多细胞模型中分析复合物。因此，我们的目标是研究体内功能 EXOSC3 中独特的致病氨基酸取代的后果与一系列轻度疾病有关 PCH1b 中的严重表型。我们生成了一系列与 EXOSC3 疾病相关的错义等位基因 通过 CRISPR/Cas9 编辑技术对果蝇直系同源物 Rrp40 进行突变。我们之前在苍蝇方面的工作 揭示了神经元对 Rrp40 的需求增强。此外，我们对大脑富集的RNA-seq分析 Rrp40突变体的转录组显示功能重要神经元的稳态水平增加 转录本，表明果蝇 RNA 外泌体亚基 Rrp40 中引起疾病​​的氨基酸变化 导致神经元功能障碍。我们现在的目标是描述致病氨基酸替代是如何引起疾病的 Rrp40 改变果蝇体内神经系统发育的分子和细胞景观。我们 将检验 RNA 外泌体调节 RNA 的假设，这些 RNA 对正常的正常运转至关重要。 神经发育和功能，其中一个独特的子集由 RNA 外泌体帽调节 Rrp40 亚基 (EXOSC3) 通过三个互补的目标：1) 评估氨基的功能后果 果蝇 RNA 外泌体 Rrp40 中的酸取代对应于导致 PCH1b 的酸取代； 2） 探究 Rrp40 突变如何影响果蝇内关键神经元转录物的表达和定位 脑; 3) 利用遗传筛选来确定特定 RNA 外泌体是否异常积累 目标会破坏果蝇的发育和/或体内平衡。成功完成这些目标将提供 对 RNA 外泌体生物学的协同理解和对 PCH1b 疾病病因学的宝贵见解。