Expression level of R155H mRNA in the knock-in mouse model.
Expression level of R155H mRNA in the knock-in mouse model.
复制标题
敲入小鼠模型中 R155H mRNA 的表达水平。
DOI：
10.1016/j.bbrc.2020.01.021
复制
发表时间：
2020
影响因子：
3.1
通讯作者：
Kimonis,Virginia
中科院分区：
文献类型：
--
作者：
Cheng,Cheng;Weiss,Lan;Ta,Lac;Kimonis,Virginia
我们阅读了Clemen等人的文章，标题为“杂合R155 C VCP突变：对人类有毒！对老鼠无害？”[1]饶有兴趣在这篇文章中，作者的目的是建立IBMPFD模型，通过产生一个常见的患者特异性突变R155 C的小鼠。经过一系列的行为、组织学和生化分析，他们发现R155 C杂合子小鼠没有表现出人类IBMPFD患者中发现的严重病理学。为了理解为什么R155 C VCP基因敲入没有导致表型变化，Clemen等人分析了R155 C杂合子小鼠中野生型和突变型VCP mRNA种类的量，并指出在骨骼肌和脑组织中分别仅检测到5%和7%的突变型mRNA。这一结果表明，在R155 C杂合子小鼠中，野生型VCP mRNA占主导地位，从而产生功能性VCP蛋白。先前发表的携带患者特异性突变R155 H的IBMPFD小鼠模型的结果表明，杂合R155 H小鼠显示出年龄依赖性肌无力。此外，组织学和生物化学分析表明，R155 H杂合小鼠形成集中的肌核，上调TDP 43和对TDP 43、VCP和泛素呈阳性的包涵体，重现了人类患者中表现出的表型[2]。因此，我们研究了R155 C小鼠中的mRNA发现是否也在R155 H杂合子小鼠中观察到。分析来自R155 H杂合、R155 H纯合和野生型对照小鼠的心脏和骨骼肌的mRNA以估计野生型和R155 H mRNA的量。将等量的PCR产物进行NciI限制酶消化，其中R155 H突变的存在破坏了内源性NciI位点。正如预期的那样，来自野生型小鼠的VCP mRNA被完全消化成两个较小的片段。然而，来自R155 H杂合子小鼠的VCP mRNA种类在NciI切割后基本上是完整的，与在R155 H纯合子小鼠中观察到的相似。此外，在含有野生型和纯合cDNA的等量混合物的PCR产物的NciI切割中观察到部分消化（三条带）（图1）。这些结果表明，来自R155 H杂合子小鼠的VCP mRNA可能含有大部分突变型VCP，从而引起突变型VCP蛋白和小鼠的进一步生理异常。不同的VCP突变可能会不同地影响mRNA的合成或代谢。在R155 C的情况下，VCP突变体mRNA表达的减少（或降解的增加）被推测在R155 C杂合小鼠中起保护作用。
We read the article by Clemen et al. entitled as “The heterozygous R155C VCP mutation: Toxic in humans! Harmless in mice?”[1] with great interest. In this article, authors aimed to establish IBMPFD model by generating a mouse harboring a common patient-specific mutation R155C. After a battery of behavioral, histological and biochemical analyses, they found that R155C heterozygous mice did not manifest the profound pathology found in human IBMPFD patients. To understand why R155C VCP knock-in led to no phenotypic changes, Clemen et al. analyzed the amount of wildtype and mutant VCP mRNA species in the R155C heterozygous mice, and indicated that there was only 5% and 7% mutant mRNA detected in the skeletal muscle and the brain tissue, respectively. This result suggests that in the R155C heterozygous mice, the wildtype VCP mRNA dominates, and thus produce functional VCP protein.It is intriguing to see the unequal distribution of wildtype and mutant VCP mRNA species in the R155C heterozygous mice. Previously published results in IBMPFD mouse model harboring patient-specific mutation R155H have suggested that heterozygous R155H mice display age-dependent muscle weakness. Furthermore, histology and biochemical analyses suggest that R155H heterozygous mice form centralized muscle nuclei, upregulation of TDP43 and inclusion bodies positive for TDP43, VCP and Ubiquitin, recapitulating the phenotypes manifested in human patients [2]. Therefore, we investigated whether the mRNA findings in the R155C mice was also observed in the R155H heterozygous mice. The mRNA from heart and skeletal muscle of R155H heterozygous, R155H homozygous and wildtype control mice were analyzed to estimate the amounts of wildtype and R155H mRNA. Equal amount of PCR products were subjected to NciI restriction enzyme digestion, where the presence of R155H mutation destroys the endogenous NciI site. As expected, the VCP mRNA from wildtype mice were completely digested into two smaller fragments. However, VCP mRNA species from R155H heterozygous mice were largely intact upon NciI cutting, similar to what was observed in R155H homozygous mice. Also, a partial digestion (three bands) was observed in NciI cutting of PCR product containing equal mixture of wildtype and homozygous cDNA (Fig. 1). These results indicate that the VCP mRNA from R155H heterozygous mice may contain a large portion of mutant VCP, giving rise to the mutant VCP protein and further physiological abnormality in mice. It is possible that different VCP mutations can impact mRNA synthesis or metabolism differently. In the case R155C, a decrease of VCP mutant mRNA expression (or increase in degradation) is presumed to play a protective role in the R155C heterozygous mice.