An alternative isoform of RRM2B as a genetic modifier in Huntington's disease

亨廷顿病（HD）是一种显性遗传性神经退行性疾病， 由亨廷顿蛋白（HTT）基因中的三核苷酸重复扩增引起的疾病，人们对其知之甚少， 治疗发展我们的首要目标是更好地了解HD发病机制， 通过研究HD的遗传修饰剂进行治疗干预的途径，最初由GeM-HD鉴定 财团本提案的目的是评估一种HD修饰剂，核糖核苷二磷酸还原酶 亚基M2 B（RRM 2 B），通过描述其对线粒体调节的亚型特异性生物活性， 表征其对HD发病机制的作用。我们的假设是chr 8 GWAS修饰子中的两个SNP 单倍型rs 1037699和rs 5893603显示出降低RRM 2B同种型2水平，在同种型2中起关键作用。 特异性复合物，改变总体RRM 2B活性和HD发病机制。完成后，这项工作将 验证RRM 2B同种型2作为HD发病机制的调节剂，并作为新的治疗方法的基础。 改变疾病的策略，并通过以下方式深入了解HD的基本机制：1） 表征亚型特异性RRM 2B活性，重点是其在线粒体调节中的作用，对亚细胞 LCL和人神经元祖细胞中的定位、应激反应测定和敲低实验 （hNPC）和从具有不同CAG大小的我们的新HTT等基因系列iPSC分化的神经元（17，40， 2）在应激和时间内鉴定含有RRM 2B同种型2的关键细胞蛋白复合物 使用APEX介导的定量蛋白质组学的依赖性方式; 3）通过 使用新技术，如非编码反义核酸， RNA（SIENUP）。新的技术和资源包括敏感的等位基因特异性SRM-MS，用于靶向 定量，最近优化的基因治疗工具和创新的HD细胞模型系统，旨在 精确描述与HD相关的CAG特征。该提案将揭示RRM 2B之间的接口 生物学和HD发病机制，建立RRM 2B作为靶向途径，并可能揭示其他 致病级联中的参与者。我们的期望是上调RRM 2B亚型2水平的策略将 对HD表型有积极的影响，为治疗提供了关键的候选药物。总的来说，积极的 这项工作的结果将使我们能够更好地了解遗传修饰剂对HD的影响，并验证 为合理的药物设计提供直接的靶点，为未来的治疗策略提供额外的途径。

The pathogenic mechanism of Huntington’s disease (HD), a fatal, dominantly inherited neurodegenerative disorder caused by trinucleotide repeat expansion in the huntingtin (HTT) gene, is poorly understood, stymieing therapeutic development. Our overarching goal is to better understand HD pathogenesis and identify targetable pathways for therapeutic intervention by studying genetic modifiers of HD, initially identified by the GeM-HD consortium. The objective of this proposal is to evaluate one HD modifier, ribonucleoside-diphosphate reductase subunit M2 B (RRM2B), by delineating its isoform-specific biological activity on mitochondrial regulation and characterizing its effect on HD pathogenesis. Our hypothesis is that the two SNPs in chr8 GWAS modifier haplotype, rs1037699 and rs5893603, shown to lower RRM2B isoform 2 levels, play a critical role in isoform 2- specific complex(es), altering overall RRM2B activity and HD pathogenesis. Upon completion, this work will validate RRM2B isoform 2 as a modulator of HD pathogenesis and serve as a foundation for novel therapeutic disease-altering strategies as well as providing insight into the fundamental mechanism of HD by: 1) Characterizing isoform-specific RRM2B activity, focusing on its role in mitochondrial regulation, on subcellular localization, stress-response assays, and knockdown experiments in LCLs and human neuronal progenitor cells (hNPCs) and neurons differentiated from our new HTT isogenic series of iPSCs with different CAG sizes (17, 40, 51 and 61); 2) Identifying key cellular protein complexes containing RRM2B isoform 2 in a stress and time dependent manner using APEX-mediated quantitative proteomics; 3) Evaluating potential therapeutic effects by augmenting RRM2B isoform 2 levels in HD patient cells using novel technologies, such as non-coding antisense RNA (SIENUP). Novel techniques and resources include sensitive allele-specific SRM-MS for targeted quantification, recently optimized gene therapy tools, and innovative HD cell model systems, designed to precisely delineate CAG signatures relevant for HD. This proposal will unveil the interface between RRM2B biology and HD pathogenesis, establishing RRM2B as a targetable pathway and potentially revealing other players in the pathogenic cascade. Our expectation is that strategies to upregulate RRM2B isoform 2 level will have a positive effect on HD phenotypes, providing a key candidate for therapeutics. Overall, the positive outcomes of this work will enable us to better understand the effect of genetic modifiers on HD and validate immediate targets for rational drug design, providing additional avenues for future therapeutic strategies.