Higgins Joseph J, Tal Adit L, Sun Xiaowei, Hauck Stefanie C R, Hao Jin, Kosofosky Barry E, Rajadhyaksha Anjali M
Department of Pediatrics, Division of Pediatric Neurology, York Presbyterian Hospital, Weill Cornell Medical College, New York, New York 10065, USA.
J Neurogenet. 2010 Mar;24(1):18-26. doi: 10.3109/01677060903567849.
A mild form of autosomal recessive, nonsyndromal intellectual disability (ARNSID) in humans is caused by a homozygous nonsense mutation in the cereblon gene (mutCRBN). Rodent crbn protein binds to the intracellular C-terminus of the large conductance Ca(2+)-activated K(+)channel (BK(Ca)). An mRNA variant (human SITE 2 INSERT or mouse strex) of the BK(Ca) gene (KCNMA1) that is normally expressed during embryonic development is aberrantly expressed in mutCRBN human lymphoblastoid cell lines (LCLs) as compared to wild-type (wt) LCLs. The present study analyzes the temporal and spatial distribution of crbn and kcnma1 mRNAs in the mouse brain by the quantitative real-time reverse transcriptase-polymerase chain reaction (qPCR). The spatial expression pattern of endogenous and exogenous crbn proteins is characterized by immunostaining. The results show that neocortical (CTX) crbn and kcnma1 mRNA expression increases from embryonic stages to adulthood. The strex mRNA variant is >3.5-fold higher in embryos and decreases rapidly postnatally. Mouse crbn mRNA is abundant in the cerebellum (CRBM), with less expression in the CTX, hippocampus (HC), and striatum (Str) in adult mice. The intracytoplasmic distribution of endogenous crbn protein in the mouse CRBM, CTX, HC, and Str is similar to the immunostaining pattern described previously for the BK(Ca) channel. Exogenous hemagglutinin (HA) epitope-tagged human wt- and mutCRBN proteins using cDNA transfection in HEK293T cell lines showed the same intracellular expression distribution as endogenous mouse crbn protein. The results suggest that mutCRBN may cause ARNSID by disrupting the developmental regulation of BK(Ca) in brain regions that are critical for memory and learning.
人类常染色体隐性非综合征性智力障碍（ARNSID）的一种轻度形式是由cereblon基因（mutCRBN）中的纯合无义突变引起的。啮齿动物的crbn蛋白与大电导Ca(2+)激活钾通道（BK(Ca)）的细胞内C末端结合。与野生型（wt）淋巴母细胞系（LCL）相比，BK(Ca)基因（KCNMA1）的一种mRNA变体（人类SITE 2 INSERT或小鼠strex）在mutCRBN人类LCL中异常表达，该变体通常在胚胎发育期间表达。本研究通过定量实时逆转录聚合酶链反应（qPCR）分析了小鼠脑中crbn和kcnma1 mRNA的时空分布。通过免疫染色对内源性和外源性crbn蛋白的空间表达模式进行了表征。结果表明，新皮质（CTX）中crbn和kcnma1 mRNA的表达从胚胎期到成年期增加。strex mRNA变体在胚胎中高出>3.5倍，出生后迅速下降。成年小鼠中，小鼠crbn mRNA在小脑（CRBM）中丰富，在CTX、海马体（HC）和纹状体（Str）中的表达较少。小鼠CRBM、CTX、HC和Str中内源性crbn蛋白的胞质分布与先前描述的BK(Ca)通道的免疫染色模式相似。在HEK293T细胞系中使用cDNA转染外源性血凝素（HA）表位标记的人类wt-和mutCRBN蛋白，显示出与内源性小鼠crbn蛋白相同的细胞内表达分布。结果表明，mutCRBN可能通过破坏对记忆和学习至关重要的脑区中BK(Ca)的发育调节而导致ARNSID。
