Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.
Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.
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DOI：
10.1093/brain/awad249
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发表时间：
2023-12-01
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影响因子：
--
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中科院分区：
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RNA聚合酶III(Pol III)相关性低髓鞘性白质营养不良(POLR3-HLD)，又称4H白质营养不良，是一种以髓鞘减少、牙齿减少、性腺激素减退为主要特征的严重神经退行性疾病。POLR3-HLD是由POLR3亚基编码基因的双等位致病变异引起的。虽然大约一半的患者携带编码RNA聚合酶III亚单位B的POLR3B突变，但没有基于该POLIII亚单位突变的脑白质营养不良的体内模型。在这里，我们确定了POLR3BΔ10(Δ10)对人类细胞POL III的影响，并利用小鼠Δ10的同源突变建立了一种可诱导/条件性白质营养不良的小鼠模型。采用亲和纯化-质谱法和免疫印迹技术对Pol III功能障碍的分子机制进行了研究。出生后用他莫昔芬诱导Δ10在三重转基因α-Cre/ert、R26-Stopfl-EYFP、Polr3bf1小鼠中的表达。用显微CT、体外MRI、免疫荧光、免疫组织化学、光谱共聚焦反射显微镜和免疫印迹等多种技术表征中枢和非中枢神经系统的特征。进行谱系追踪和基于谱系特异性和/或增殖标记物的联合标记的少突胶质细胞亚群动态的时间序列分析。蛋白质组学表明，Δ10导致POLR3BΔ10在人类细胞中的组装缺陷，而蛋白质印迹显示POLR3B POLR3B在人类细胞的胞浆和细胞核中的表达减少。在小鼠中，出生后依赖PDGFRα的同源突变蛋白的表达导致了隐性表型，包括严重的髓鞘减少导致共济失调、震颤、癫痫发作和有限的生存，以及牙齿减少和颅面畸形。用经典的方法定量髓鞘碱性蛋白和脂类等髓鞘成分，证实和表征了下髓鞘作用，结果与基于髓鞘膜物理性质的现代方法定量髓鞘的结果一致。谱系追踪揭示了低髓鞘表型的潜在机制：少突胶质前体增殖和分化缺陷导致在出生后髓鞘形成过程中无法产生足够数量的成熟少突胶质细胞。总之，我们确定了Polr3bΔ10突变的特征，并开发了一种动物模型，该模型概括了由POLR3B突变引起的POLR3-HLD的特征，揭示了疾病的发病机制，并为治疗干预措施的发展打开了大门。Micell-Robinson等人。提出了第一个基于Polr3b突变的脑白质营养不良小鼠模型，Polr3b是一种编码RNA聚合酶III(PolIII)亚单位的基因。该模型中疾病的临床表现，包括髓鞘减少、牙齿减少和头面部异常，与POLR3B突变引起的脑白质营养不良患者相似。
RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions. Michell-Robinson et al. present the first leukodystrophy mouse model based on mutation of Polr3b, a gene encoding a subunit of RNA polymerase III (Pol III). The clinical manifestations of disease in the model, including hypomyelination, hypodontia and craniofacial abnormalities, resemble those seen in patients with leukodystrophy caused by POLR3B mutations.
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影响因子：
2.8
作者：
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通讯作者：
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影响因子：
16.2
作者：
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影响因子：
3.3
作者：
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影响因子：
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作者：
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通讯作者：
Bernard, Genevieve
DOI：
10.1002/cpz1.89
发表时间：
2021-05
期刊：
Current protocols
影响因子：
--
作者：
Dobson ETA;Cimini B;Klemm AH;Wählby C;Carpenter AE;Eliceiri KW
通讯作者：
Eliceiri KW