In situ hybridization study of the distribution of choline acetyltransferase mRNA and its splice variants in the mouse brain and spinal cord
In situ hybridization study of the distribution of choline acetyltransferase mRNA and its splice variants in the mouse brain and spinal cord
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DOI：
10.1016/j.neuroscience.2008.12.054
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发表时间：
2009-03
期刊：
影响因子：
3.3
通讯作者：
S. Trifonov;T. Houtani;S. Hamada;M. Kase;M. Maruyama;T. Sugimoto
中科院分区：
文献类型：
--
作者：
S. Trifonov;T. Houtani;S. Hamada;M. Kase;M. Maruyama;T. Sugimoto
胆碱乙酰转移酶是催化神经递质乙酰胆碱生物合成的酶。胆碱乙酰转移酶的七种不同类型的5‘-非编码区的mRNA是从不同启动子区域的胆碱能基因座转录而来的，并在小鼠体内通过选择性剪接产生。采用地高辛标记的核探针和原位杂交组织化学方法，观察N1、R1、R2、R3、R4和总胆碱乙酰转移酶基因在小鼠中枢神经系统的表达。胆碱乙酰转移酶转录本的相对水平在成熟的胆碱能神经系统的不同细分中有很大的不同。在中枢神经系统的一些预期区域中，发现了与所研究的所有五个剪接变体(R1、R2、R3、R4和N1)以及与共同蛋白质编码区的核探针杂交的神经元。它们包括基底前脑、纹状体、桥中脑被盖、脑干的运动和自主神经核团以及脊髓。R1和R2剪接变异体表达水平中等到极高的神经元分布在前脑和脑干核团中。另一方面，R3、R4和N1剪接变异体在脑干运动和自主神经核团以及脊髓腹角和侧角有中到高水平的表达，而在前脑胆碱能结构中表达水平较低。在大脑皮质、海马和内侧缰核的神经元中未检测到N1、R1、R2、R3和R4剪接变异体的表达。使用共同蛋白编码区的核探针，可以高水平标记内侧缰核的神经元，而低水平标记皮质内的神经元。海马区未见总胆碱乙酰转移酶基因的显著杂交。这些结果有力地表明：(1)R1和R2是在前脑核团神经元中表达的主要剪接变体；(2)R1、R2、R3、R4和N1剪接变体在脑干运动、自主神经核和脊髓腹角和侧角的表达几乎相同；(3)从缺乏其他亚型推断，M型胆碱乙酰转移酶是一种剪接变体，主要在大脑皮层和缰核内侧表达。
Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of the neurotransmitter acetylcholine. Seven types of mRNA for choline acetyltransferase that differ in the 5′-noncoding region are transcribed from the cholinergic gene locus from different promoter regions and produced by alternative splicing in the mouse. Digoxigenin-labeled riboprobes and in situ hybridization histochemistry were used to investigate the expression of N1, R1, R2, R3, R4 and total choline acetyltransferase mRNA in the mouse CNS. The relative levels of choline acetyltransferase transcripts differed dramatically in distinct subdivisions of the mature cholinergic nervous system. Neurons hybridizing with antisense riboprobes for all of the five investigated splice variants (R1, R2, R3, R4 and N1) as well as those hybridizing with riboprobe for the common protein-coding region were found in a number of expected regions in the CNS. They include the basal forebrain, striatum, pontomesencephalic tegmentum, motor and autonomic nuclei of the brainstem, and spinal cord. Neurons with a moderate to very high level of expression of R1 and R2 splice variants were distributed in both the forebrain and brainstem nuclei. On the other hand, R3, R4 and N1 splice variants revealed a moderate to high level of expression in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord compared to a low expression level in forebrain cholinergic structures. No expression of the N1, R1, R2, R3 and R4 splice variants was detectable in the neurons of the cerebral cortex, hippocampus and medial habenular nucleus. With the riboprobe for the common protein-coding region, the neurons of the medial habenular nucleus could be labeled at high level, while intrinsic cortical neurons were labeled at low level. Hippocampus revealed no significant hybridization for total choline acetyltransferase mRNA. These findings strongly suggested that: (1) R1 and R2 were the major splice variants expressed in the neurons of forebrain nuclei; (2) R1, R2, R3, R4 and N1 splice variants were almost equally expressed in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord; (3) inferring from a paucity of other isoforms, M type choline acetyltransferase mRNA is a splice variant predominantly expressed in the cerebral cortex and medial habenular nucleus.