A humanized mouse model for UBQLN2-associated ALS-dementia

摘要 该R21提案的目标是开发一种人源化的小鼠模型，用于UBQLN2相关的 肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)，毁灭性的神经退行性疾病 位于临床疾病谱的两端。在20多种不同的基因中， 泛素(Ub)伴侣UBQLN2的ALS/FTD，X连锁突变导致不同的神经学 表型从伴有或不伴有FTD的单纯ALS到原发性侧索硬化症和痉挛截瘫。 UBQLN2的ALS/FTD相关突变破坏了其折叠并促进了其聚集；然而， UBQLN2突变引发ALS/FTD神经变性的机制尚不清楚。UBQLn2 突变的转基因啮齿动物重现了UBQLN2的组织病理学，但表现出高度不同 神经学表型，从明显的运动神经元变性到看不见的表型。一个 对这些研究和转基因啮齿动物模型的警告是，野生型的过度表达 UBQLN2也会引起毒性，可能是通过破坏Ub的动态平衡。另一方面，虽然在那里 迫切需要在内源性水平上表达罪魁祸首ALS突变的模型，小鼠 携带ALS相关UBQLN2敲门突变的人表现出弱的表型，排除了机制 学习。对于人类神经退行性疾病的小鼠模型来说，这一直是一个普遍的问题。 为了在内源表达水平上增强UBQLN2的毒性，我们小组开发了一种 UBQLN2组合突变体(UBQLN24XALS)含有四种不同的临床突变，可降低其 以半相加的方式溶解。UBQLN24XALS与野生型UBQLN2相比毒性增强 或UBQLN2临床突变体在果蝇中过表达或从UBQLN2中表达时 IPS来源运动神经元中的Knockin等位基因。UBQLN24XALS毒性的遗传抑制因子 果蝇也抑制了其对iMN的毒性，提示UBQLN24XALS的毒性机制至少是 在苍蝇和哺乳动物中部分保守。在这里，我们建议生成一个有条件的、人性化的 UBQLN24XALS小鼠模型允许在时间和空间上控制UBQLN24XALS的小鼠神经 系统。在对神经退行性变表型进行充分评估后，将使用UBQLN24XALS小鼠作为工具 使用组学方法和从同源基因收集的信息破译ALS疾病的途径 果蝇和ALS-UBQLN细胞模型的研究学习到的信息也应该告知 肌萎缩侧索硬化症/肌萎缩侧索硬化症是由零星和其他遗传原因引起的。该提案的目标是：1) 获得条件的、人源化的UBQLN2WT到UBQLN24XALS基因开关小鼠；2)组织病理学和 Nestin(Nes)-Cre，UBQLN24XALS小鼠的行为学评估。

Abstract The objective of this R21 proposal is to develop a humanized mouse model for UBQLN2-associated amyotrophic sclerosis (ALS) and frontotemporal dementia (FTD), devastating neurodegenerative disorders that lie on either end of a clinical disease spectrum. Among more than 20 different genes implicated in ALS/FTD, X-linked mutations in the ubiquitin (Ub) chaperone UBQLN2 cause diverse neurologic phenotypes, ranging from pure ALS with or without FTD, to primary lateral sclerosis and spastic paraplegia. ALS/FTD-associated mutations in UBQLN2 disrupt its folding and promote its aggregation; however, the mechanisms whereby UBQLN2 mutations instigate neurodegeneration in ALS/FTD are unclear. UBQLN2 mutant transgenic rodents recapitulated UBQLN2 histopathology but manifested highly discrepant neurological phenotypes ranging from patent motor neuron degeneration to no observable phenotype. A caveat to these studies—and transgenic rodent models in general—is that overexpression of wild-type UBQLN2 also elicits toxicity, likely through disruption of Ub homeostasis. On the other hand, while there is a strong need for models in which culprit ALS mutations are expressed at endogenous levels, mice harboring ALS-associated UBQLN2 knockin mutations exhibit weak phenotypes, precluding mechanistic studies. This has been a general problem for mouse models of human neurodegenerative diseases. In an attempt to augment UBQLN2 toxicity at endogenous expression levels, our group developed a combinatorial UBQLN2 mutant (UBQLN24XALS) harboring four different clinical mutations that reduce its solubility in a semi-additive manner. UBQLN24XALS caused enhanced toxicity relative to wild-type UBQLN2 or UBQLN2 clinical mutants when overexpressed in Drosophila or when expressed from a UBQLN2 knockin allele in iPS-derived motor neurons (iMNs). Genetic suppressors of UBQLN24XALS toxicity in Drosophila also suppressed its toxicity in iMNs suggesting that UBQLN24XALS toxicity mechanism is at least partially conserved in flies and mammals. Here, we propose to generate a conditional, humanized UBQLN24XALS mouse model to allow spatially and temporally controlled UBQLN24XALS in the mouse nervous system. After a full evaluation of neurodegenerative phenotypes, UBQLN24XALS mice will be used as a tool to decipher ALS disease pathways using ‘omics approaches and information gleaned from orthologous studies in Drosophila and cellular models of ALS-UBQLN2. Information learned should also inform ALS/FTD arising sporadically and from other genetic causes. The objectives of the proposal are to: 1) Generate conditional, humanized UBQLN2WT to UBQLN24XALS gene switch mice; 2) Histopathologic and behavioral assessment of Nestin (Nes)-Cre, UBQLN24XALS mice.