Quantitative localization of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phenotypes in normal and Rett syndrome brain by laser scanning cytometry
Quantitative localization of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phenotypes in normal and Rett syndrome brain by laser scanning cytometry
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DOI：
10.1093/hmg/10.17.1729
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发表时间：
2001-08-15
影响因子：
3.5
通讯作者：
Greco, CM
中科院分区：
文献类型：
--
作者：
LaSalle, JM;Goldstine, J;Greco, CM
Rett 综合征 (RTT) 是一种 X 连锁显性神经发育障碍，由编码甲基 CpG 结合蛋白 2 (MeCP2) 的 MECP2 突变引起。 RTT 发病机制中的一个主要悖论是，普遍转录的 MECP2 的突变如何导致出生后发育过程中中枢神经系统 (CNS) 特有的表型。为了解决这个问题，我们使用了一种新方法，通过免疫荧光和激光扫描细胞术原位定量野生型和突变型 MeCP2 的水平和分布。令人惊讶的是，在正常脑中观察到MeCP2表达水平的细胞异质性，其中一部分细胞表现出高表达(MeCP2(hl))，而其余细胞表现出低表达(MeCP2(lo))。通过对多个组织阵列上 MeCP2 的自动定量，与人类和小鼠的非 CNS 组织相比，MeCP2 在 CNS 中的表达显着更高。正常人脑中 MeCP2 表达表型的定量定位显示出马赛克但不同的分布模式，MeCP2(hi) 神经元在大脑第四层最高，MeCP2(lo) 神经元在小脑颗粒层最高。在女性 RTT 大脑中，MECP2 突变体表达细胞被鉴定为 MeCP2 C 末端表位阴性的细胞。 MECP2 突变体表达细胞随机定位于 Rett 大脑和小脑，并显示出正常的 MeCP2 表达和 N 端特异性抗 MeCP2。这些结果证明了 MeCP2 高表达的 CNS 特异性细胞表型，并表明 RTT 中的 MECP2 突变仅在 MeCP2(hi) 细胞中表现出来。此外，我们的结果证明了激光扫描细胞术在检查疾病发病机制中复杂细胞表型方面的能力。
Rett syndrome (RTT) is an X-linked, dominant neurodevelopmental disorder caused by mutations in MECP2, encoding the methyl-CpG-binding protein 2 (MeCP2). A major paradox in the pathogenesis of RTT is how mutations in ubiquitously transcribed MECP2 result in a phenotype specific to the central nervous system (CNS) during postnatal development. To address this question, we have used a novel approach for quantitating the level and distribution of wild-type and mutant MeCP2 in situ by immunofluorescence and laser scanning cytometry. Surprisingly, cellular heterogeneity in MeCP2 expression level was observed in normal brain with a subpopulation of cells exhibiting high expression (MeCP2(hl)) and the remainder exhibiting low expression (MeCP2(lo)). MeCP2 expression was significantly higher in CNS compared with non-CNS tissues of human and mouse by automated quantitation of MeCP2 on multiple tissue arrays. Quantitative localization of MeCP2 expression phenotypes in normal human brain showed a mosaic, but distinct, distribution pattern, with MeCP2(hi) neurons highest in layer IV of the cerebrum and MeCP2(lo) neurons highest in the granular layer of the cerebellum. In female RTT brains, MECP2 mutant-expressing cells were identified as cells negative for the MeCP2 C-terminal epitope. MECP2 mutant-expressing cells were randomly localized in Rett cerebrum and cerebellum and showed normal MeCP2 expression with N-terminal-specific anti-MeCP2. These results demonstrate a CNS-specific cellular phenotype of MeCP2 high expression and suggest that MECP2 mutations in RTT are only manifested in MeCP2(hi) cells. In addition, our results demonstrate the power of laser scanning cytometry in examining complex cellular phenotypes in disease pathogenesis.