■纤毛缺陷会导致与突变负荷相关的异质性表型,从而导致发育受损。与野生型(WT)幼犬相比,先前报道的Man1a2基因纯合缺失会导致新生幼犬的致死性呼吸衰竭和肺损伤降低。杂合突变的影响,救援的可能性尚不清楚。
■我们假设生存率和肺损伤,(a)与WT相比,Man1a2+/-杂合子和Man1a2-/-空新生幼崽逐渐减少,和(b)可以通过用N-乙酰半胱氨酸(NAC)进行妊娠治疗来增强,抗氧化剂.
■Man1a2+/-成年小鼠从繁殖前一周开始通过妊娠喂养NAC或安慰剂。监测新生幼崽的存活24小时。肺,收集肝脏和尾巴的形态,基因分型,和转录分析。
■生存(p=0.0001,Kaplan-Meier)和肺损伤百分比(p=0.0001,ANOVA)通过Arl13b+呼吸道上皮细胞的频率逐渐减少,如假设。与安慰剂相比,妊娠NAC治疗增强了(a)每种基因型幼崽的肺损伤,(b)在杂合幼崽中存活(p=0.017),但在WT或无效幼崽中不存活。肺而不是肝的整个转录组显示了在活的杂合子和WT幼崽中相同的上调和下调基因的模式,与死的杂合幼崽和空幼崽完全相反。系统生物学分析能够重建蛋白质相互作用网络,从而产生功能相关的模块及其相互作用。在这些网络中,突变的Man1a2酶有助于肺发育必需的蛋白质的异常合成。相关的未折叠蛋白质,NAC可以减轻低氧和氧化应激反应。与发育中的人胎儿肺转录组的比较表明,NAC可能在Man1a2突变小鼠中恢复正常的血管和上皮管形态发生。
■Man1a2突变小鼠的生存和肺损伤,其与N-乙酰半胱氨酸的改善是基因型依赖性的。NAC介导的拯救取决于氧化和低氧应激在调节发育过程中的纤毛功能和器官发生中的核心作用。
BACKGROUND: Ciliary defects cause heterogenous phenotypes related to mutation burden which lead to impaired development. A previously reported homozygous deletion in the Man1a2 gene causes lethal respiratory failure in newborn pups and decreased lung ciliation compared with wild type (WT) pups. The effects of heterozygous mutation, and the potential for rescue are not known.
OBJECTIVE: We hypothesized that survival and lung ciliation, (a) would decrease progressively in Man1a2 +/- heterozygous and Man1a2 -/- null newborn pups compared with WT, and (b) could be enhanced by gestational treatment with N-Acetyl-cysteine (NAC), an antioxidant.
METHODS: Man1a2+/- adult mice were fed NAC or placebo from a week before breeding through gestation. Survival of newborn pups was monitored for 24 h. Lungs, liver and tails were harvested for morphology, genotyping, and transcriptional profiling.
RESULTS: Survival (p = 0.0001, Kaplan-Meier) and percent lung ciliation (p = 0.0001, ANOVA) measured by frequency of Arl13b+ respiratory epithelial cells decreased progressively, as hypothesized. Compared with placebo, gestational NAC treatment enhanced (a) lung ciliation in pups with each genotype, (b) survival in heterozygous pups (p = 0.017) but not in WT or null pups. Whole transcriptome of lung but not liver demonstrated patterns of up- and down-regulated genes that were identical in living heterozygous and WT pups, and completely opposite to those in dead heterozygous and null pups. Systems biology analysis enabled reconstruction of protein interaction networks that yielded functionally relevant modules and their interactions. In these networks, the mutant Man1a2 enzyme contributes to abnormal synthesis of proteins essential for lung development. The associated unfolded protein, hypoxic and oxidative stress responses can be mitigated with NAC. Comparisons with the developing human fetal lung transcriptome show that NAC likely restores normal vascular and epithelial tube morphogenesis in Man1a2 mutant mice.
CONCLUSIONS: Survival and lung ciliation in the Man1a2 mutant mouse, and its improvement with N-Acetyl cysteine is genotype-dependent. NAC-mediated rescue depends on the central role for oxidative and hypoxic stress in regulating ciliary function and organogenesis during development.