Mechanistic studies and therapeutics for ALS/FTD linked to UBQLN2 mutations

摘要 UBQLN2突变导致伴有额颞部的肌萎缩侧索硬化症X连锁显性遗传 痴呆(ALS/FTD)。编码的泛素-2蛋白(UBQLN2)属于一个保守的小家族。 通过结合和处理泛素化的蛋白质来维持蛋白稳定的蛋白质 蛋白酶体和自噬-溶酶体降解系统。越来越多的证据表明， 通过干扰这两个清除系统中的任何一个而产生的蛋白抑制会导致神经退化。因此， 了解UBQLN2蛋白的功能和功能障碍对神经退行性变具有广泛的意义 疾病。UBQLN2突变导致发病的机制正在出现。朝向这个方向 目标，我们为P497S和P506T UBQLN2突变建立了转基因(TG)小鼠模型，表明 这两个突变株都概括了人类疾病的中心特征，包括UBQLN2的沉积 包涵体、认知障碍、运动神经元疾病和TDP-43病理学。通过蛋白质组学和 免疫印迹分析发现，P497S突变小鼠在参与P497S基因突变的蛋白质中发生了重大变化 自噬和线粒体健康所需的蛋白质中。使用一种新型的报告系统，与 UBQLN2基因敲除(KO)细胞，我们发现UBQLN2中的ALS/FTD连锁突变通过阻断 自噬小体酸化。我们将这一缺陷与UBQLN2在调节细胞周期中的一个新功能联系在一起 液泡型(H+)-ATPase泵。从脊髓中提纯的线粒体的呼吸分析显示突变 UBQLN2动物的氧化磷酸化水平随着年龄的增长而下降。类似的缺陷也被发现在 UBQLN2KO细胞，提示线粒体活性的功能下降可能源于UBQLN2的丢失 功能。支持这一观点的是，我们发现野生型(WT)UBQLN2挽救了线粒体的功能 而携带ALS/FTD突变的UBQLN2则没有。此外，我们还发现WT UBQLn2是 需要并调节线粒体蛋白输入，而ALS/FTD突变体UBQLN2蛋白缺乏 活动的一部分。对于这次更新，我们提出了4个目标，它们将利用这些令人兴奋的发现，以及 在上一个供资期间取得的其他重要调查结果。在目标1中，我们将确定分子 UBQLN2在空泡-ATPase调节中的作用机制及ALS/FTD UBQLN2的原因 突变的蛋白质在活性上被破坏。在目标2中，我们将确定潜在的分子机制 UBQLN2在线粒体蛋白输入和活性方面的功能和功能障碍。目标3是测试 过表达UBQLN1可减轻SOD1小鼠ALS模型的疾病。目标4是找回这两个人 来自冷冻精子库的突变UBQLN2小鼠品系，以获得更好的表型。这项研究的结果是 可能会大大提高我们对UBQLN2蛋白在健康中发挥作用的机制的了解 和疾病，其教训不仅可以用于ALS/FTD的治疗干预，而且 其他类似蛋白代谢缺陷的神经退行性疾病。

Summary UBQLN2 mutations cause X-linked dominant inheritance of amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). The encoded ubiquilin-2 protein (UBQLN2) belongs to a small family of conserved proteins that function to maintain proteostasis by binding and disposing ubiquitinated proteins through the proteasome and autophagy-lysosomal degradation systems. There is increasing evidence that disruption in proteostasis by interference in either of these clearing systems cause neurodegeneration. Therefore, understanding how UBQLN2 proteins function and dysfunction has broad implications for neurodegenerative diseases. The mechanisms by which UBQLN2 mutations cause pathogenesis are emerging. Towards this goal, we generated transgenic (Tg) mouse models for the P497S and P506T UBQLN2 mutations, showing that both mutant lines recapitulate central features of the human disease, including deposition of UBQLN2 inclusions, cognitive deficits, motor neuron disease and TDP-43 pathology. Through proteomic and immunoblot analysis we found that P497S mutant mice have major alterations in proteins involved in autophagy and in proteins required for mitochondrial health. Using a novel reporter system in combination with UBQLN2 knockout (KO) cells, we found ALS/FTD-linked mutations in UBQLN2 impede autophagy by blocking autophagosome acidification. We tied the defect to a novel function of UBQLN2 in regulation of the vacuolar(H+)-ATPase pump. Respiration assays of mitochondria purified from the spinal cord revealed mutant UBQLN2 animals have an age-dependent decline in oxidative phosphorylation. Similar defects were found in UBQLN2 KO cells, suggesting the functional decline in mitochondrial activity may stem from loss of UBQLN2 function. In support of this idea, we found that wild type (WT) UBQLN2 rescued the mitochondrial function deficits whereas UBQLN2 bearing an ALS/FTD mutation did not. Furthermore, we found WT UBQLN2 is required and regulates mitochondrial protein import whereas ALS/FTD mutant UBQLN2 proteins are deficient of the activity. For this renewal, we propose 4 aims that will capitalize on these exciting discoveries, as well as other important findings made during the last funding period. In Aim 1 we will determine the molecular mechanisms by which UBQLN2 functions in vacuolar-ATPase regulation and clarify why ALS/FTD UBQLN2 mutant proteins are disrupted in the activity. In Aim 2 we will determine the molecular mechanisms underlying UBQLN2 function and dysfunction in mitochondrial protein import and activity. Aim 3 is to test whether overexpression of UBQLN1 alleviates disease in SOD1 mouse models of ALS. Aim 4 is to recover the two mutant UBQLN2 mouse lines from sperm cryo-stocks for better phenotypes. The results of this research are likely to considerably advance our knowledge of the mechanisms by which UBQLN2 proteins function in health and disease, the lessons of which could be exploited for therapeutic intervention in not only ALS/FTD, but other neurodegenerative disease where similar proteostasis defects may operate.