A humanized mouse model for UBQLN2-associated ALS-dementia

摘要 该R21提案的目的是开发用于UBQLN 2相关的人源化小鼠模型。 肌萎缩性硬化症（ALS）和额颞叶痴呆（FTD），破坏性神经退行性疾病 位于临床疾病谱两端。在20多个不同的基因中， ALS/FTD，泛素（Ub）伴侣UBQLN 2中的X连锁突变导致不同的神经系统疾病 表型，范围从单纯的ALS伴或不伴FTD，到原发性侧索硬化症和痉挛性截瘫。 UBQLN 2中ALS/FTD相关突变破坏其折叠并促进其聚集;然而， UBQLN 2突变引起ALS/FTD神经变性的机制尚不清楚。UBQLN 2 突变转基因啮齿类动物重现了UBQLN 2组织病理学，但表现出高度差异， 神经系统表型从明显的运动神经元变性到没有可观察到的表型。一 对这些研究和一般转基因啮齿动物模型的警告是野生型 UBQLN 2也可能通过破坏Ub体内平衡而增强毒性。另一方面，在 强烈需要一种模型，其中罪魁祸首ALS突变在内源性水平表达，小鼠 携带ALS相关的UBQLN 2敲入突变表现出弱表型，排除了机制性 问题研究这是人类神经退行性疾病小鼠模型的普遍问题。 为了在内源性表达水平上增加UBQLN 2的毒性，我们的研究小组开发了一种 组合UBQLN 2突变体（UBQLN 24 XALS）携带四种不同的临床突变， 以半加和的方式溶解。相对于野生型UBQLN 2，UBQLN 24 XALS引起增强的毒性 或UBQLN 2临床突变体，当在果蝇中过表达时或当从UBQLN 2表达时 iPS衍生的运动神经元（iMN）中的敲入等位基因。UBQLN 24 XALS毒性的遗传抑制因子 果蝇也抑制了其在iMN中的毒性，表明UBQLN 24 XALS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 UBQLN 24 XALS小鼠模型，以允许在小鼠神经系统中空间和时间控制UBQLN 24 XALS 系统在对神经退行性表型进行全面评估后，将使用UBQLN 24 XALS小鼠作为工具 使用组学方法和从骨科收集的信息来解读ALS疾病途径， 果蝇和ALS-UBQLN 2细胞模型的研究。了解的信息还应告知 ALS/FTD偶发性和由其他遗传原因引起。该提案的目标是：1） 产生有条件的人源化UBQLN 2 WT至UBQLN 24 XALS基因转换小鼠; 2）组织学和免疫组织化学分析; Nestin（内斯）-Cre，UBQLN 24 XALS小鼠的行为评估。

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.