Lee Wing C, Tsoi Yuen K, Dickey Chad A, Delucia Michael W, Dickson Dennis W, Eckman Christopher B
Department of Pharmacology, Mayo Clinic College of Medicine, Jacksonville, FL 32224, USA.
Neurobiol Dis. 2006 Aug;23(2):273-80. doi: 10.1016/j.nbd.2006.03.005. Epub 2006 Jun 8.
The twitcher mouse is a pathologically and enzymatically authentic model of globoid cell leukodystrophy (GLD, Krabbe disease) that has been widely used for the evaluation of potential therapeutic approaches. This naturally occurring mouse model contains a premature stop codon (W339X) in the galactosylceramidase (GALC) gene that abolishes enzymatic activity. Using either immunocytochemical approaches or Western blot methodology, we have been unable to detect the truncated form of GALC expected to be produced in these animals. Nonsense-mediated mRNA decay (NMD) is a cellular protection mechanism that degrades newly synthesized transcripts containing a premature termination codon (PTC). Since the naturally occurring mutation in the twitcher mouse introduces a PTC, we hypothesized that NMD might affect the degradation of GALC mRNA in these animals. Consistent with this hypothesis, we determined that the amount of GALC transcript was inversely proportional to the number of twitcher containing alleles. Similar reductions in GALC mRNA were detected in a twitcher-derived Schwann cell line (TwS1) when compared to wild-type Schwann cells (IMS32). Anisomycin, emetine and puromycin, inhibitors of NMD, effectively increased the level of GALC transcript in the TwS1 cells providing further support for nonsense-mediated mRNA decay being the mechanism by which no GALC protein is detected in these animals. Understanding the mechanistic differences between the lack of enzymatic activity in the twitcher model and that observed with the missense mutations that cause human disease yields not only novel therapeutic insights but also highlights the need for additional animal models.
颤抖小鼠是球状细胞脑白质营养不良（GLD，克拉伯病）在病理和酶学方面真实的模型，已被广泛用于评估潜在的治疗方法。这种自然发生的小鼠模型在半乳糖神经酰胺酶（GALC）基因中含有一个过早的终止密码子（W339X），该密码子消除了酶活性。使用免疫细胞化学方法或蛋白质印迹法，我们均未能检测到预期在这些动物中产生的截短形式的GALC。无义介导的mRNA降解（NMD）是一种细胞保护机制，可降解含有过早终止密码子（PTC）的新合成转录本。由于颤抖小鼠中的自然突变引入了PTC，我们推测NMD可能会影响这些动物中GALC mRNA的降解。与该假设一致，我们确定GALC转录本的量与含颤抖等位基因的数量成反比。与野生型雪旺细胞（IMS32）相比，在源自颤抖小鼠的雪旺细胞系（TwS1）中检测到GALC mRNA有类似程度的减少。NMD抑制剂茴香霉素、依米丁和嘌呤霉素有效提高了TwS1细胞中GALC转录本的水平，这为无义介导的mRNA降解是这些动物中未检测到GALC蛋白的机制提供了进一步支持。了解颤抖小鼠模型中酶活性缺乏与导致人类疾病的错义突变中观察到的酶活性缺乏之间的机制差异，不仅能产生新的治疗见解，还凸显了对其他动物模型的需求。
