Two new mouse models of Gjb1-associated Charcot-Marie-Tooth disease type 1X.
Two new mouse models of Gjb1-associated Charcot-Marie-Tooth disease type 1X.
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Gjb1 相关 1X 型腓骨肌萎缩症的两种新小鼠模型。
DOI：
10.1111/jns.12588
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Burgess,RobertW
中科院分区：
文献类型：
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作者：
Tadenev,ALD;Hatton,CL;Pattavina,B;Mullins,T;Schneider,R;Bogdanik,LP;Burgess,RobertW
背景 1X 型腓骨肌萎缩症是由 GJB1 突变引起的，GJB1 是与遗传性周围神经病相关的第二个最常见基因。 GJB1 基因编码连接蛋白 32 (CX32)，这是一种在髓鞘神经胶质细胞中表达的间隙连接蛋白。该基因是 X 连锁的，突变会导致功能丧失。目的已鉴定出大量与疾病相关的变异，其中许多会导致蛋白质的错误运输和错误定位。现有的缺乏 Gjb1 表达的敲除小鼠提供了有效的 CMT1X 动物模型，但完全缺乏蛋白质可能无法完全重现异常 CX32 蛋白引起的疾病机制。为了更好地代表人类CMT1X相关突变谱，我们生成了一个新的Gjb1knockin小鼠模型。方法使用CRISPR/Cas9基因组编辑来产生Gjb1中携带R15Q突变的小鼠。此外，我们还发现了密码子 7 (del2) 中具有早期移码突变的第二个等位基因。使用临床相关的分子、组织学、神经生理学和行为测定对小鼠进行分析。结果两个等位基因均产生可通过免疫荧光在雪旺细胞中检测到的蛋白质，其中一些蛋白质正确定位于朗飞结。然而，这两种等位基因也会导致周围神经病变，伴有薄髓鞘和脱髓鞘轴突，以及轴突退化和再生，主要发生在远端运动神经中。神经传导速度在晚年仅轻度降低，复合肌肉动作电位振幅并未降低。两个等位基因的血浆中神经丝轻链水平均升高。 del2 小鼠在约 3 个月大时发病，而 R15Q 小鼠在 5-6 个月大时发病较晚，表明功能丧失较轻。两种等位基因在神经肌肉性能的加速转棒和握力测试中的表现与野生型同窝小鼠相当。解释我们已经生成并表征了两种新的 CMT1X 小鼠模型，这将有助于未来的机制和临床前研究。
BackgroundCharcot–Marie–Tooth disease type 1X is caused by mutations inGJB1, which is the second most common gene associated with inherited peripheral neuropathy. TheGJB1gene encodes connexin 32 (CX32), a gap junction protein expressed in myelinating glial cells. The gene is X‐linked, and the mutations cause a loss of function.AimsA large number of disease‐associated variants have been identified, and many result in mistrafficking and mislocalization of the protein. An existing knockout mouse lackingGjb1expression provides a valid animal model of CMT1X, but the complete lack of protein may not fully recapitulate the disease mechanisms caused by aberrant CX32 proteins. To better represent the spectrum of human CMT1X‐associated mutations, we have generated a newGjb1knockin mouse model.MethodsCRISPR/Cas9 genome editing was used to produce mice carrying the R15Q mutation inGjb1. In addition, we identified a second allele with an early frame shift mutation in codon 7 (del2). Mice were analyzed using clinically relevant molecular, histological, neurophysiological, and behavioral assays.ResultsBoth alleles produce protein detectable by immunofluorescence in Schwann cells, with some protein properly localizing to nodes of Ranvier. However, both alleles also result in peripheral neuropathy with thinly myelinated and demyelinated axons, as well as degenerating and regenerating axons, predominantly in distal motor nerves. Nerve conduction velocities were only mildly reduced at later ages and compound muscle action potential amplitudes were not reduced. Levels of neurofilament light chain in plasma were elevated in both alleles. The del2 mice have an onset at ~3 months of age, whereas the R15Q mice had a later onset at 5–6 months of age, suggesting a milder loss of function. Both alleles performed comparably to wild type littermates in accelerating rotarod and grip strength tests of neuromuscular performance.InterpretationWe have generated and characterized two new mouse models of CMT1X that will be useful for future mechanistic and preclinical studies.